Polyvalent conjugate vaccine for cancer

ABSTRACT

This invention provides a polyvalent vaccine comprising at least two conjugated antigens selected from a group containing glycolipid antigen, polysaccharide antigen, mucin antigen, glycosylated mucin antigen and an appropriate adjuvant. This invention also provides a multivalent vaccine comprising at least two of the following: glycosylated MUC-1-32mer, Globo H, GM2, Le y , Tn(c), sTN(c), and TF(c). This invention provides the vaccine above, wherein the adjuvant is saponin-based adjuvant. This invention provides a method for inducing immune response in a subject comprising administering an effective amount of the vaccine above to the subject. Finally, this invention provides a method for treating cancer in a subject comprising administering an appropriate amount of the vaccine above to the subject.

The application disclosed herein is a continuation-in-part ofInternational Application No. PCT/US02/21348, filed Jul. 5, 2002, whichclaims priority of U.S. Ser. No. 60/303,494, filed on Jul. 6, 2001 andU.S. Ser. No. 60/347,231, filed on Jan. 10, 2002, the contents of whichare hereby incorporated by reference into this application.

The invention disclosed herein was made with United States governmentsupport under NIH Grant Nos. CA33049 and CA52477 from the United StatesDepartment of Health and Human Services. Accordingly, the United StatesGovernment has certain rights in this invention.

Throughout this application, various references are referred to.Disclosures of these publications in their entireties are herebyincorporated by reference into this application to more fully describethe state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

Tumor-specific antigens have been identified and pursued as targets forvaccines. Previous work from the inventors' has shown that monovalentvaccines utilizing the tumor antigens Globo H, Lewis^(y), GM2,glycosylated MUC-1, Tn(c), sTn(c), or TF(c) conjugated to KLH to be safewith local erythema and edema but minimal systemic toxicities. As aresult of vaccination with these monovalent vaccines, most patientsgenerated specific high titer IgM or IgG antibodies against therespective antigen-KLH conjugates. The present invention provides apolyvalent vaccine wherein the components of the monovalent vaccines arecombined and administered with an adjuvant as treatment for cancer.

SUMMARY OF THE INVENTION

The invention disclosed herein provides a polyvalent vaccine comprisingat least two conjugated antigens selected from a group containingglycolipid antigen, polysaccharide antigen, mucin antigen, glycosylatedmucin antigen and an appropriate adjuvant. This invention also providesthe multivalent vaccine, comprising glycosylated MUC-1-32 mer, Globo H,GM2, Le^(y), Tn(c), and TF(c). This vaccine may comprise glycosylatedMUC-1-G5, Globo H, GM2, Le^(y), Tn(c), sTN(c), and TF(c). This inventionprovides the vaccine above, wherein the adjuvant is saponin-basedadjuvant.

This invention also provides a method for inducing immune response in asubject comprising administering an effective amount of the vaccineabove to the subject. Finally, this invention provides a method fortreating cancer in a subject comprising administering an appropriateamount of the vaccine above to the subject.

DETAILED DESCRIPTION OF THE INVENTION

The invention disclosed herein provides a polyvalent vaccine comprisingat least two conjugated antigens selected from a group containingglycolipid antigen, polysaccharide antigen, mucin antigen, glycosylatedmucin antigen and an appropriate adjuvant.

The glycolipid includes but is not limited to Globo H, a Lewis antigenand a ganglioside. The Lewis antigen includes but is not limited toLe^(y) and sialyl Le^(a). The ganglioside includes fucosylated GM1, GM2,GD2, or GD3. In another embodiment, the mucin is a MUC peptide. In afurther embodiment, the MUC peptide is MUC-1, MUC-2 or MUC-16. Thepolysaccharide antigen includes but is not limited to Tn(c), sTn(c),TF(c), and polysialic acid.

This invention provides a bivalent, trivalent, tetravalent, pentavalent,hexavalent, and heptavalent vaccine. The vaccine comprises at least twoconjugated antigens selected from a group containing glycolipid antigen,polysaccharide antigen, mucin antigen, glycosylated mucin antigen and anappropriate adjuvant.

In an embodiment, the hexavalent vaccine comprises glycosylated MUC-1-32mer, Globo H, GM2, Le^(y), Tn(c), and TF(c). In a further embodiment,the range of MUC-1-32 mer is from about 0.1 to 30 ug. In yet anotherembodiment, the range of Globo H is from about 0.1 to 100 ug. In still afurther embodiment, the range of GM2 is from about 0.1 to 100 ug. In anadditional embodiment, the range of Le^(y) is from about 0.1 to 60 ug.In a further embodiment, the range of Tn(c) is from about 0.1 to 100 ug.In an additional embodiment, the range of TF(c) is from about 0.1 to 30ug.

In a separate embodiment, the adjuvant is saponin based. The adjuvantincludes QS21 and GPI-0100. In an embodiment, the range of QS21 is fromabout 25 to about 200 ug. In another embodiment, QS21 is about 100 ug.In a separate embodiment, the adjuvant is GPI-0100 with a range fromabout 1 to 25 mg. In an embodiment, GPI-0100 is about 10 mg.

This invention provides a heptavalent vaccine comprising at least twoconjugated antigens selected from a group containing glycolipid antigen,polysaccharide antigen, mucin antigen, and glycosylated mucin antigenand an appropriate adjuvant. In an embodiment, the vaccine comprisesglycosylated MUC-1-G5, Globo H, GM2, Le^(y), Tn(c), sTN(c), and TF(c).In another embodiment, the range of MUC-1-G5 is from about 0.1 to 30 ug.In a further embodiment, the range of Globo H is from about 0.1 to 100ug. In another embodiment, the range of GM2 is from about 0.1 to 100 ug.In still another embodiment, the range of Le^(y) is from about 0.1 to 60ug. In an embodiment, the range of Tn(c) is from about 0.1 to 100 ug. Ina further embodiment, the range of sTn(c) is from about 0.1 to 100 ug.In yet another embodiment, the range of TF(c) is from about 0.1 to 30ug.

This invention provides the vaccine above, wherein the adjuvant issaponin-based adjuvant. These saponin-based adjuvants include but arenot limited to QS21 and GPI-0100.

In an embodiment, the range of QS21 is from about 25 to 200 ug. Inanother embodiment, the QS21 is about 10 ug. In a separate embodiment,the adjuvant is GPI-0100 and the range is from about 1 to 25 mg. In apreferred embodiment, GPI-0100 is about 10 mg.

This invention provides a polyvalent vaccine comprising a conjugatedglycosylated antigen, a conjugated ganglioside antigen and anappropriate adjuvant, wherein the antigens are conjugated to a carrier.In an embodiment, the carrier is Keyhole Limpet Hemocyanin (KLH).

This invention provides the polyvalent vaccine above comprising at leasttwo conjugated antigens selected from a group containing glycolipidantigen, polysaccharide antigen, mucin antigen, and glycosylated mucinantigen and an appropriate adjuvant for cancer. In an embodiment, thecancer is prostate, breast or ovarian cancer.

This invention also provides a method for inducing immune response in asubject comprising administering an effective amount of the abovevaccine to the subject.

Furthermore, this invention provides a method for treating cancer in asubject comprising administering an appropriate amount of the abovevaccine to the subject.

This invention also provides a composition comprising the above vaccineand a carrier.

This invention also provides a pharmaceutical composition comprising theabove vaccine and a pharmaceutically acceptable carrier.

In addition, the invention provides a vaccine for small cell lung cancercomprising at least two conjugated antigens selected from the groupcontaining Globo H, fucosylated GM1, GM2, GD2, GD3, sialyl Le^(a) andpolysialic acid. This invention also provides a method for inducingimmune response in a subject bearing small cell lung cancer comprisingadministering an effective amount of the above vaccine to the subject.This invention furthermore provides a method for treating a subjectbearing small cell lung cancer comprising administering an effectiveamount of the above vaccine to the subject.

In addition, this invention provides the above vaccine, furthercomprising an antigen selected from a group containing CA125, or aportion thereof, KSA peptide or protein, and PSMA, or a portion thereof.

This invention includes the above vaccines which further comprise otherantigens which can induce antibody and/or immune response. Asillustrated throughout the specification, the antigen used may bemodified to increase its immunogenicity. Said antigens include but arenot limited to CA125, or a portion thereof, KSA peptide or protein, andPSMA, or a portion thereof. As can be easily appreciated by the ordinaryskilled artisan, only a portion of the antigen may be required forinduction of immune response from a subject.

As stated herein, subjects are organisms which have immune response. Thesubject includes but is not limited to humans. Said subject could bedomestic animals, such as dogs and cats.

This invention further provides the above compositions and apharmaceutically acceptable carrier, thereby forming pharmaceuticalcompositions.

This invention also provides a pharmaceutical composition comprising acombination as described above and a pharmaceutically acceptablecarrier. For the purposes of this invention, “pharmaceuticallyacceptable carriers” means any of the standard pharmaceutical carriers.Examples of suitable carriers are well known in the art and may include,but are not limited to, any of the standard pharmaceutical carriers suchas a phosphate buffered saline solution and various wetting agents.Other carriers may include additives used in tablets, granules andcapsules, etc. Typically such carriers contain excipients such asstarch, milk, sugar, certain types of clay, gelatin, stearic acid orsalts thereof, magnesium or calcium stearate, talc, vegetable fats oroils, gum, glycols or other known excipients. Such carriers may alsoinclude flavor and color additives or other ingredients. Compositionscomprising such carriers are formulated by well-known conventionalmethods.

The invention will be better understood by reference to the ExperimentalDetails which follow, but those skilled in the art will readilyappreciate that the specific experiments detailed are only illustrative,and are not meant to limit the invention as described herein, which isdefined by the claims which follow thereafter.

EXPERIMENTAL DETAILS First Series of Experiments

Polyvalent (Hexavalent) Conjugate Vaccine for Prostate, Breast, Ovarianand Small Cell Lung Cancer

Tumor-specific antigens have been identified and pursued as targets forvaccines. The inventors' previous work has shown that monovalentvaccines utilizing the tumor antigens Globo H, Lewis^(y), GM2,glycosylated MUC-1, Tn(c), or TF(c) conjugated to KLH to be safe withlocal erythema and edema but minimal systemic toxicities. As a result ofvaccination with these monovalent vaccines, most patients generatedspecific high titer IgM or IgG antibodies against the respectiveantigen-KLH conjugates. The present invention provides a hexavalentvaccine wherein the components of the monovalent vaccines are combinedand administered with an adjuvant as treatment for prostate, breast,ovarian and small cell lung cancer.

A vaccine consisting of a unique combination of six tumor antigensadministered with a saponin immunological adjuvant QS-21 or GPI-0100.The antigens are glycosylated MUC-1-32 mer, Globo H, GM2, Le^(y), Tn(c),and TF(c). In each case the antigen is conjugated to Keyhole LimpetHemocyanin (KLH).

The preferred ranges of the antigen and adjuvant doses are as follows:

-   -   Glycosylated MUC-1-32 mer: 0.1 to 30 μg;    -   Globo H: 0.1 to 100 μg;    -   GM2: 0.1 to 100 μg;    -   Le^(y): 0.1 to 60 μg;    -   Tn(c): 0.1 to 10 μg;    -   TF(c): 0.1 to 30 μg;    -   QS-21: 100 μg;    -   GPI-0100: 1 or 25 mg.

EXAMPLE 1 A Phase I Multivalent Conjugate Vaccine Trial for Patientswith Biochemically Relapsed Prostate Cancer

-   1.0 PROTOCOL SUMMARY-   2.0 OBJECTIVE-   3.0 BACKGROUND AND RATIONALE-   4.0 VACCINE PREPARATION-   5.0 IMMUNIZATION SCHEDULE-   6.0 PRE- AND POST-THERAPY EVALUATION-   7.0 RESPONSE CRITERIA-   8.0 BIOSTATISTICAL CONSIDERATIONS-   9.0 REFERENCES    1.0 Protocol Summary:

This is a phase I pilot trial designed to assess safety using amultivalent conjugate vaccine. This trial is based on the results ofeight dose-seeking phase I monovalent glycoprotein and carbohydrateconjugate vaccine trials which have been shown to be consistentlyimmunogenic in man. These trails also allowed us to screen candidateantigens for their ability to generate high titer specific antibodiesagainst the immunizing antigen. This vaccine will consist of the highestdose of synthetic glycoprotein and carbohydrate antigens shown to elicithigh titer IgM and IgG antibodies in patients with biochemicallyrelapsed prostate cancer. The inventors' previous work has shown themonovalent vaccines to be safe with local erythema and edema but minimalsystemic toxicities. Among the antigens to be included in themultivalent vaccine are carbohydrate antigens Globo H and GM2 and theglycoprotein antigens glycosylated MUC-1-32 mer, Lewisy, Tn(c), andTF(c). The patient populations to be targeted are those patients whohave failed primary therapies such as prostatectomy or radiation or havebeen on intermittent hormonal therapy and have remained hormonallysensitive in the absence of radiographic disease. These populations musthave as the sole indication of disease progression, a rising PSA. Theinventors' data from approximately 160 men who participated in earliermonovalent vaccine trials against the aforementioned antigens have shownthat a treatment effect in the form of a decline in PSA log slopescompared with pretreatment values could be seen in patients with minimaltumor burden. A phase III double blind randomized trial with two hundredforty patients is planned based on the safety data accrued form thisproposed phase I trial. The primary endpoint of the study will be theability to assess the safety of the vaccine and the humoral response toa multivalent conjugate. Secondary endpoints will be to evaluatepost-therapy changes in PSA.

2.0 Objectives:

2.1 The primary endpoints of the study are:

2.1.1 To determine the safety of a multivalent conjugate vaccine inpatients with prostate cancer who have biochemically relapsed followingprimary therapies such as surgery or radiation.

2.1.2 Measure the antibody response against the individual components ofthe vaccine and to correlate the response to subsequent clinical course.

2.2. The secondary endpoints will be:

2.2.1 To assess post-immunization changes in prostate specific antigenlevels and other objective parameters of disease (radionuclide bone scanand/or measurable disease if present.

3.0 Background and Rationale:

3.1 Prostate cancer: Over 180,000 cases of prostate cancer will bediagnosed in the United States in 2000.¹ Of these, 30-35% will presentwith tumors beyond the confines of the gland, while an additional 25%will develop metastases in the course of the disease despite localtherapies. In these cases, a rising PSA antedates the development ofovert metastases by a median of 12-24 months. Androgen ablation is thestandard treatment with upwards of 70% of cases showing a normalizationof an abnormal PSA after therapy. When to initiate treatment remains anarea of controversy and there is no evidence that deferring therapycompromises outcomes. This observation, coupled with the fact that mostpatients relapsed within a median of 12-18 months², and that most mencan not tolerate the side effects of castration including impotency,weight gain and hot flashes, has led to the search for alternativetherapies. One such approach involves enhancing the body's own immunesystem as a means to treat local disease and prevent diseaseprogression. PSA monitoring allows the identification of patients withlow-volume disease, in whom an immunostimulatory approach may be moreefficacious relative to a heavily pretreated, symptomatic populationwith large tumor burdens. Vaccinations represent a safe interventionwith minimal toxicities that can be given as an adjuvant to surgery orradiation therapy in men at risk for systemic relapse. They can also beoffered to men with minimal tumor burdens who are progressing and whoare not willing to accept toxicities of hormonal therapy orchemotherapy. Because hormonal status may effect antigen expression andregulation, we propose to enroll patients with different hormonesensitivities. This will include patients who have not received hormonaltherapy or have been on intermittent hormonal therapy.

3.2 PSA as an endpoint for clinical trials: The availability of serumPSA determinations provides a unique trial design for testing newtherapies rapidly as changes in PSA levels over time correlate well withclinical outcomes. ³ This relationship holds for both hormone-naïve andhormonally relapsed disease. Once sequential elevations in PSA aredocumented in the setting of castrate testosterone levels, clinicalsymptoms develop in a median of 3-6 months. This observation justifiestreatment in the setting of rising PSA values, using post-therapychanges in PSA as the outcome measure. With this design, therapeuticapproaches that do not produce a defined degree of decline in PSA onmultiple determinations for a defined duration (vide infra) are notevaluated further.²

3.3 Immunologic approaches: Augmentation of the immune response tocancer can be attempted by two basic approaches: non-specificimmunopotentiation which constitutes the bulk of past and currentefforts at cancer immunotherapy, and specific immunization which has notreally been evaluated in the treatment of cancer but has contributedmuch to the control of infectious diseases. It is the knowledge ofmicrobial antigens which has permitted the development of successfulspecific immunization against infections. The lack of availability ofwell-defined human cancer antigens, on the other hand, has preventedexploration of specific immunization in the context of cancer as itshould be explored, using vaccines of defined cancer-restrictedantigenicity and demonstrating their immunogenicity in cancer patients.

3.3.1 The role of carbohydrates and mucins in prostate cancer:Carbohydrate antigens have proven to be clinically relevant and (asidefrom vaccines against toxins) are the only defined bacterial antigensused in vaccines against bacterial pathogens. Immunization withcarbohydrate antigens has also resulted in directed antibody responsesagainst human tumor cells (reviewed in ⁴), presumably because theseantibodies are known to mediate antibody-dependent cell-mediated andcomplement mediated lysis of tumor cells, complement-inducedinflammation, and phagocytosis by the reticulo-endothelial system. Theinventors' previous study in prostate cancer focused on defining theantigens expressed on the surface of prostate cancer cells.

3.3.2 Results with eight monovalent phase I trials using glycoproteinpeptides and carbohydrate antigens. Immunohistochemistry usingwell-defined monoclonal antibodies against glycoprotein and carbohydrateantigens have shown that primary and metastatic prostate carcinomaspecimens express these heretofore unknown antigens and that thesemolecules can serve as targets for immune recognition. We have studiedtwo mucin peptide antigens, MUC-1 and MUC-2 conjugated to KLH and givenwith the immune adjuvant, QS21 in the phase I setting as a doseescalating trial with 10, 30 100 and 3 μg. Patients received fivesubcutaneous vaccines over the course of twenty-six weeks at weeks 1, 2,3, 7, and 19. Twenty patients were treated in the MUC-1-KLH-QS21 trialand fifteen were treated with MUC-2-KLH-QS21 trial. All patientsdeveloped high titer IgM and IgG antibodies specific for the immunizingpeptide. Antibody titers rose by week 7 and declined usually by week 19,the time of the fifth and final vaccine. Unexpectedly, a treatmenteffect was observed after the vaccine trial was completed in the form ofa declining PSA log slope compared with pretreatment values inapproximately two-thirds of patients. In many patients, the slope beganto show a decline by week 38 with subsequent declines by week 60. Theinitial decline corresponded to the rise of antibodies following thelast immunization received at week 19. Five patients who were treatedwith the MUC-1-KLH conjugate in 1996 continue to have stable PSA logslopes without radiographic evidence of disease. Patients who weretreated with MUC-2-KLH conjugate also demonstrated a similar treatmenteffect, however, the trial has not as yet reached maturity. The vaccineswere found to be safe with erythema, tenderness and edema at theinjection site. No evidence of autoimmunity or systemic toxicity wasobserved.

3.3.3 Experience with glycolipid and carbohydrate antigens. Eighteenpatients have undergone immunization with Globo H, a glycolipid antigenexpressed on prostate cancer cells. This is the first purely syntheticcomplex carbohydrate antigen used for immunization in man capable ofgenerating high titer specific antibodies (median peak titer 1:320, IgGmedian titer 160) capable of mediating complement lysis of tumor cells.Several patients generated IgM antibody titers of 1:20, 480). Patientswere immunized with 10, 30 100 or 3 μg of Globo H-KLH plus QS21 overtwenty-six weeks. Of the patients immunized with this vaccine, sixremain active with stable PSA log slopes and no radiologic evidence ofevidence of disease over the last 2½ years. This vaccine was found to besafe with no evidence of systemic toxicity. Ganglioside antigens (acidicglycosphingolipids expressing sialic acid at one end and ceramide at theother) was also investigated in a trial comparing the immunogenicity ofhigher doses of QS21. Using GM2-KLH at 30 μg, a dose previouslyestablished in melanoma trials, 18 patients were immunized with eitherthe GM2 conjugate plus QS21 at the standard dose of 100 μg or QS21 at225 μg. Because of its potential for systemic toxicity, the lattervaccine was given as three separate immunizations to three separatesites as GM2-KLH at 10 μg plus QS21 at 75 μg subcutaneously. Nodifference in antibody titers were observed in two groups of patients;although two patients from the group given the higher dose of QS21experienced grade II myalgias. Several patients also exhibited a declinein PSA log slopes but there did not appear to be any difference betweengroups with regard to treatment effects.

4.0 Vaccine Preparation

Globo H, Lewis^(y), Tn(c), TF(c) are synthesized in the laboratory ofBio-Organic Chemistry headed by Dr. Samuel Danishefsky. MUC-1-32 mer issynthesized in the Core Peptide Synthesis Facility of The RockefellerResearch Laboratories under the aegis of Dr. Paul Tempst. It isglycosylated with Tn by Dr. Henrik Clausen at the University ofCopenhagen, Copenhagen, Denmark. GM2 is extracted from rabbit brains byProgenics, Inc., Tarrytown, N.Y.

4.1 Globo H, MUC-1-32 mer, GM2, Lewis^(y), Tn(c) and TF(c)-KLHconjugation: The above antigens will be covalently attached to KLH inDr. Livingston's laboratory. Antigen-KLH ratios between 150/1 and 800/1assuming a KLH molecular weight of 5×10⁶ will be accepted. Gels will beperformed and western blot analysis will be conducted with each lot ofantigen-KLH for comparison to future lots. Sterility and safety testingwith each lot plus QS21, at >50 times the dose/meter² to be used inclinical trials will be performed. No growth in culture and no adversereaction in mice or Guinea pigs (including weight loss of 10% or more)will be tolerated. Two or more mice will be immunized with eachantigen-KLH batch on 2-3 occasions at 1-2 week intervals and postimmunization sera tested. Antibody titers of 1/200 or greater againstantigen and 1/40 by IA or FACS staining of >25% of antigen positivecells will be accepted as proof that the construct has the appropriateimmunogenicity.

4.2 Antigen Doses: Based on previous vaccine trials in prostate cancerpatients, the following doses have been established for the multivalenttrial: glycosylated MUC-1-32 mer, 3 μg; Globo H, 10 μg; GM2, 10 μg;Le^(y), 10 μg; Tn(c), 3 μg; and TF(c), 3 μg. QS21 will be used at 100 μgas no significant difference in immunogenicity was observed with dosesas high as 225 μg.

4.3 Safety testing: Samples from the materials are sent for sterilityand safety testing. Immunogenicity of the individualpeptides/carbohydrates have been previously confirmed in mice.

5.0 Immunization Schedule:

5.1 Patient selection: All patients with evidence of biochemical relapsewill be considered. Hormonal status will be recorded on the basis ofserum testosterone levels as follows: Patients who have progressed afterprimary surgery or radiation (with or without neo-adjuvant androgenablation) who have non-castrate levels of testosterone (>50 ng/ml) willbe eligible.

5.2 Interval: The immunization schedule that we will utilize was derivedfrom the inventors' studies with other glycoprotein and carbohydrateconjugate vaccines in patients with melanoma, colon and breast cancers.

5.3 Treatment schedule and dose: Fifteen patients will be treated withspecified doses of each carbohydrate or peptide constituent as has beendetermined previously based on earlier monovalent trials completed. QS21will be administered at the standard dose of 100 ug. Sites: The vaccineconjugate will be administered subcutaneously to random sites on theupper arms and upper legs.

5.4 Dose modifications: If a patient experiences a Grade III or greaterlocal or Grade II or greater systemic toxicity at any time a decrease by50% in all components of future vaccinations will be administered forthat patient.

6.0 Pre- and Post-Therapy Evaluation:

6.1 Outcomes: The study evaluation will include parameters to assess thesafety of the vaccine, antitumor effect, as well as assessments ofimmune function. Interval safety assessments will include the PatientDiary. An overall antitumor assessment will be performed during weeks 13and 26. If the patient has not demonstrated progression of disease atweek 13 or 26, he will continue on protocol. Upon completion of thetrial, he will be monitored every 3 months with bloodwork and imagingstudies for the next 2 years or until disease progression.

6.2 Safety and Antitumor Effects:

STUDY WEEK O^(a,c) 1 2 3 7 9 13 19 26 Clinical: Performance Status X^(b) — — X X X X X  X^(h) Interval Hx & PE  X^(b) — — X X X X — X CBC,Diff, Plt. X — — X X — X — X CMP^(d), LDH X — — X — — X — X Uric acid,Phosphorus X — — X — — X — X Prothrombin time X — — — — — — — — PSA, Ac.Phos X — — X X — X — X Testosterone X — — — — — — — — U/A X — — X X X X— X Stool guaiac X — — — X — — — X Pathology Review^(e) X — — — — — — —— Imaging:^(f) Chest X-ray X — — — — — X — X Bone scan X — — — — — X — XCT Scan or MRI X — — — — — X — X Overall response — — — — — — X — Xassessments^(g) Consent for Pathologic X Correlates^(i) ^(a)Baselinestudies prior to immunization. ^(b)Within 7 days of the firstimmunization. ^(c)Within 15 days of starting treatment for biochemicalstudies; 30 days for imaging studies. Includes total bilirubin, SGOT,LDH, Alkaline Phosphatase, Creatinine, BUN. ^(d)CMP Includes totalbilirubin, SGOT, ALT, Sodium, Potassium, Chloride, CO₂, Calcium,Glucose, Total Protein, Abumin, Alkaline Phosphatase, Creatinine, BUN.^(e)Patients will be asked to obtain tissue blocks from previousdiagnostic/therapeutic procedures will be obtained and the patient'stumor evaluated for the presence of the antigens byimmunohistochemistry. The presence of any antigen on paraffin materialis not a criterion for entry and no biopsy procedures will be performedspecifically for enrollment. ^(f)Abdominal and pelvic CT scans with andwithout contrast, chest x-ray and any other tests deemed necessary todocument evaluable disease. ^(g)Overall response assessment includes therepetition of abnormal imaging and biochemical studies used to assessdisease, and in selected cases, immune function. ^(h)Repeat at 3 monthintervals for 2 years or until disease progression is documented.^(i)Patients will be asked to sign a separate consent for pathologiccorrelative studies under IRB [90-40: Dr. H. Scher, P. I. - Molecularcorrelations in human prostate cancer].6.3 Immune Function:

STUDY WEEK 0 1 2 3 7 9 13 19 26 VACCINATION* — 1 2 3 4 — — 5 — B-CELLTESTING — 1 2 3 4 5 6 7 8 *No skin tests will be performed as previoustrials indicated that there is minimal or no reactivity with intradermaladministration of the antigens studied.

Antibody response: Peripheral blood (30 cc) will be drawn prior tovaccine immediately before each vaccination, as well as weeks 9, 13, and26 to assess B-cell function. Thereafter, blood will be drawn at 3-monthintervals (up to one year from the first vaccination), or as long asdetectable immunity against the antigens persist. Depending on theantibody response, additional testing involving proliferation andcloning may be performed at a later date. The patients' sera will betested by ELISA for antibodies against purified antigens as well as avariety of cell lines expressing (or not) the antigens included in thevaccine.

7.0 Response Criteria:

7.1 Patients WITHOUT bi-dimensionally measurable disease are evaluableby post-therapy changes in PSA as follows:

7.1.1 Complete Response (CR): Normalization of the PSA (<1.0 or 2.0 asdefined in 4.1.1) for 3 successive evaluations at least 2 weeks apart.

7.1.2 Partial Response (PR): Decrease in PSA value by >50% abovebaseline (without normalization) for 3 successive evaluations.

7.1.3 Stabilization (STAB): Patients who do not meet the criteria for PRor PROG for at least 90 days will be considered stable.

7.1.4 Progression (PROG): Three consecutive increases in PSA, to >50%above baseline.

7.2 Duration of response: Non-measurable disease: Time from initiationof therapy until a 50% increase from the PSA nadir value is documentedon three successive determinations.

8.0 Biostatistical Consideration

8.1 This is an exploratory study to study the safety of a multivalentconjugate vaccine which will be taken to phase III clinical trials.Patients with prostate cancer who have experienced a PSA recurrenceafter radical prostatectomy or radiation therapy are eligible. Allfifteen patients will receive the same dose. The dose is based onprevious vaccine trials in prostate cancer patients, the following doseshave been established for the multivalent trial: glycosylated MUC-1-32mer, 3 μg; Globo H, 10 μg; GM2, 10 μg; Le^(y), 10 μg; Tn(c), 3 μg; andTF(c), 3 μg. QS21 will be used at 100 μg as no significant difference inimmunogenicity was observed with doses as high as 225 μg. Subjects willbe followed for two years or until the development of metastaticdisease. Bone scan and CT scans (or MRI where clinically appropriate)will be performed approximately at week 13, 26, and approximately every3 months thereafter until the development of metastatic disease. Inaddition, PSA measurements will be obtained at weeks 3, 7, 13, 26, andevery approximately 3 months thereafter in order to study the effect ofthe vaccine on the probability of developing metastatic disease and theeffect of the vaccine on PSA slope over time, respectively.²⁴

8.2 In order to be eligible for the study, patients must have a risingPSA following radical prostatectomy or radiation therapy. This detectionof PSA following treatment must occur within two years. Using the ASTROdefinition, three consecutive PSA rises are considered a biochemicalfailure after radical prostatectomy or radiation therapy. The date offailure should be the midpoint between the postsurgical (orpostirradiation) nadir PSA and the first of the three consecutiverises.²⁵ In addition, patients must have a PSA doubling time (DT) lessthan 5 months. PSA doubling time is determined prior to treatment and isequal to ln(2) divided by the least squares derived slope of log PSAover time (log PSA slope >0.15).²⁶ The time interval in which PSA DTwill be based will consist of a minimum of three PSA measurements in atwelve-month interval prior to randomization. Patients who meet thisrequirement are considered at a higher risk for metastatic disease andwill be eligible for this trial.

8.3 The primary objective of this study is to determine the safety andthe humoral response of the multivalent vaccine in preparation for thephase III trial. The primary endpoint will be the time to radiographicprogression of disease. The secondary objective is to study the rate ofchange in PSA over time.

REFERENCES

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Gendler S, Lancaster C, Taylor-Papadimitriou J, Duhig T, et al:    Molecular cloning and expresion of human tumor-associated    polymorphic epithelial mucin. J Biol Chem 265:15286-15293, 1990.-   8. Gendler S J, Spicer A P, Lalani E-N, et al: Structure and biology    of a carcinoma-associated mucin, MUC1. Am Rev Res, 144:S42-S47,    1991.-   9. Carrato C, Balague C, De Bolos C, Gonzalez E, Gambus G, et al:    Differential apomucin expression in normal and neoplastic human    gastrointestinal tissues. Gastroenterology, 107:160-172, 1994.-   10. Ho S B, Niehans G A, Lyftogt C, Yan P s, et al: Heterogeneity of    mucin gene expression in normal and neoplastic tissues. Cancer Res,    53:641-651, 1993.-   11. Slovin, S. F., Livingston, P., Zhang, S., Keeperman, K., Mhatre,    S., Adluri, S., Ghossein, R., Glazewski, J., Gordils, J.,    Danishefsy, S., and Scher, H. I.: Targeted therapy in prostate    cancer (PC): vaccination with a glycoprotein, MUC-1-KLH-QS21 peptide    conjugate. Proc Am Soc Clin Onc., 16:311a (Abstr 1107), 1997.-   12. Slovin, S. F., Livingston, P., Keeperman, K., Mhatre, S.,    Danishefsky, S., Glazewski, J., Adluri, S., Zhang, S., Gordils, J.,    and Scher, H. I.: Targeted therapy in prostate cancer: vaccination    with glycoprotein, MUC-1-KLH-QS21 peptide conjugate. J. Urol.,    157:160 (Abstr 620), 1997.-   13. Zing P X, Apostolopoulos V, Prenzoska J, et al: Petioled binding    sites recognized by anti-mucin (MUC-2) antibodies. Scand J Immun,    591-592, 1994.-   14. Slovin, S. F., Ragupathi, G., Donaldson, C., Olkiewicz, K.,    Terry, K., DePaolo, R., Livingston, P. O, and Scher, H. I.:    MUC-2-KLH conjugate vaccine: Immunogenicity in patients with    relapsed prostate cancer. Proc. Amer. Assoc. Cancer Res,    40:312(Abstr#2071), 1999.-   15. Ragugpathi, G., Adluri, R., Amaravathi, R., Howarad, L.,    Gilewski, T., Slovin S. F., and Livingston, P. O.: Specificity    analysis of sera from breast and prostate cancer patients vaccinated    with MUC1-KLH and MUC-2-KLH conjugate vaccines. Proc. Amer. Assoc.    Cancer Res, 40:312(Abstr #2070), 1999-   16. Slovin, S. F., Livingston, P. O., Danishefsky, S., Mhatre, S.,    Ragupathi, R., Depaolo R., Sames, D., Terry K., Bauso, A., Kelly, W.    K., Fazzari, M., and Scher, H. I.: Carbohydrate vaccines as    immunotherapy for prostate cancer (PC): Globo-H-KLH conjugate plus    QS21. Proc Am Soc Clin Onc, 17:433a (Abstr 1669), 1998.-   17. Slovin, SF, Ragupathi G, Adluri S, Ungers G, Terry K, Kim S,    Spassova M, Bornmann, W G, Fazzari M, Dantis L, Olkiewicz K, Lloyd K    O, Livingston P O, Danishefsky S J, and Scher H I: Carbohydrate    vaccines in cancer: Immunogenicity of a fully synthetic globo    hexasaccharide conjugate in man. Proc Natl Acad Sci, USA,    96:5710-5715, 1999.-   18. Slovin, S., Ragupathi, G., Israel, R., Terry, K., Bauso, A.,    Fazzari, M., Kelly, K., Reyes, S., Livingston, P., and Scher, H.    Ganglioside vaccines in relapsed prostate cancer (PC): Experience    with GM2-KLE conjugate plus the immunologic adjuvant, QS21-A trial    comparing QS21 doses. Proc. Amer. Soc. Clin. Onc., 18:316Aa(Abstr    #1214), 1999.-   19. Livingston P O, Natoli E J, Calves M J: Vaccines containing    purified GM2 ganglioside elicit antibodies in melanoma patients.    Proc natl Acad Sci USA, 27:537, 1987 (Abstract).-   20. Slovin, S F, et al: Tn-cluster (c)-KLH/PAM vaccine conjugates in    biochemically relapsed prostate cancer (PC): Phase I trial results.    Proc. Amer. Assoc. Cancer Res, in press.-   21. MacLean G D, Reddish M, Koganty, R R, Wong T, Gandhi S,    Smolenski M, Samuel J, Nabholtz J M, and Longenecker B M:    Immunization of breast cancer patients using a synthetic sialyl-Tn    glycoconjugate plus Detox adjuvant. Cancer Immunol Immunotherap    36:215-222, 1993.-   22. Zhang S, Walberg L A, Ogata S, Itzkowitz S H, Koganty R R,    Reddish M, Gandhi S S, Longenecker B M, Lloyd K O and Livingston P    O: Immune sera and monoclonal antibodies define two configurations    for the sialyl Tn tumor antigen. Cancer Res 55:3364-3368, 1995.-   23. MacLean G D, Bowen-Yacyshyn M B, Samuel J, Meikle A, Stuart G,    Nation J, Poppema S, Jerry M, Koganty R, Wong T, and Longenecker B    M: Active immunization of human ovarian cancer patients against a    common carcinoma (Thompson-Friedenreich) determinant using a    synthetic carbohydrate antigen. J Immunotherapy, 11:292-305, 1992-   24. Scher, H. I, Slovin, S. F., Kelly, W. K., Livingston, P. O.,    Danishefsky, S., Fazzari, M., Terry, K. And Heller, Glen:    Intermediate markers in assessing response to vaccine therapies.    Proc Am Soc Clin Onc, 17:324a (Abstr 1247), 1998.-   25. Patel, A, Dorey, F, Franklin, J, and DeKernion, J B: Recurrence    pattterns after radical retropubic prostatectomy: Clinical    usefulness of prostate specific antigen doubling times and log slope    prostate specific antigen. J Urol, 158:1441-1445, 1997.-   26. O'Brien, P C, Fleming, T R: A multiple testing procedure for    clinical trials. Biometrics 35:549-556, 1979.

EXAMPLE 2 Hexavalent Vaccine Immunogenicity Trial in Mice Methods

Serological Analyses

1. ELISA (Enzyme-Linked Immunosorbent Assay):

ELISA assays were performed as described below. Antigen in ethanol or in0.1 M carbonate buffer (pH 11) were coated on ELISA plates at 0.2μg/well for glycolipids and 0.1 μg/well for peptides. Serially dilutedantiserum was added to each well and alkaline phosphatase-conjugatedgoat anti-mouse IgM or anti-mouse IgG was added at a dilution of 1:200(Southern Biotechnology Associates, Inc, Birmingham, Ala.). Goatanti-mouse IgG and IgM conjugated with alkaline phosphatase obtainedfrom Kierkegaard and Perry Labs, (Gaithersburg, Md.) were used as secondantibodies. ELISA titer is defined as the highest dilution yielding anabsorbance of 0.1 or greater over that of normal control mouse sera.

2. Cell Surface Reactivity Determined by FACS:

The cell surface reactivity of immune sera was tested on human celllines. Single cell suspensions of 2×10⁵ cells/tube were washed in PBSwith 3% fetal calf serum (FCS) and 0.01M NaN₃ and incubated with 20 μlof 1:20 diluted sera or monoclonal antibody mAb for 30 min on ice. Aftertwo washes with 3% FCS in PBS, 20 μl of 1:15 diluted goat anti-mouse IgMor IgG-labeled with fluorescein-isothiocyanate (FITC, SouthernBiotechnology Associates Inc. Birmingham, Ala.) was added, and themixture incubated for 30 min. After a final wash, the positivepopulation and mean fluorescence intensity of stained cells weredifferentiated using FACScan, Becton & Dickinson Immunocytometry, SanJose, Calif.

APPENDIX B Hexavalent Vaccine Immunopenicity Trial in Mice Four femaleCB6F1 mice were vaccinated weekly for three weeks with Hexavalentvaccine (Hexavalent vaccine in Polyval-KLH conjugate plus 20 ug QS21 permouse). The injections were SC, at 2 Sites, with 95 ul/site. [Viallabeled Polyval in Polyval-KLH conjugate plus 100 ug QS21/1.0 ml. Totalvol. 1.0 ml. Lot #081100) Pre-vaccination sera was drawn from eachmouse. Sera was drawn again at 10 days post third vaccination. The micewere weighed prior to and post vaccination (at 24 hr post, at 48 hrpost, at one week post and at 2 weeks post). For controls, the followingmonoclonal antibodies were used:

SEROLOGY ELISA plates were coated with 0.1 ug/well of one of thefollowing antigens: GloboH-ceramide, GM2(lgM), MUC1G5, Tn-′HSA, Tf-′HSAELISA plates were coated with 0.2 ug/well of one of the followingantigens: GM2 (lgG), Ley Sera was tested at an initial dilution of 1:40,with subsequent 2-fold dilutions (with the exception of GloboH for which3-fold dilutions were used). FACS analysis was performed on two celltines: MCF7 and LSC (5 X 10⁵ cells per tube). Sera was added at a 1:20dilution (25 ul/tube). Each post 3rd vacc. sera was set against itscorresponding pre-sera (each pre-sera was set at 10%). Appendix BResults ELISA Globo H GM2 Le^(y) IgG IgM IgG IgM IgG IgM Mouse # preserapost 3rd presera post 3rd presera post 3rd presera post 3rd presera post3rd presera post 3rd 1 0 40 120 1,080 40 40 40 80 0 40 80 640 2 0 80 403,240 40 40 40 80 0 40 80 80 4 0 360 120 3,240 0 0 0 80 0 40 40 640 5 040 0 3,240 0 40 0 80 0 160 40 320 +control VK9 1:25,600 αGM2 >>>1:1,000,000 BR96 1:3,200 (1 ug/ul) MUC1G5 Tf-′HSA Tn-′HSA IgG IgM IgGIgM IgG IgM Mouse # presera post 3rd presera post 3rd presera post 3rdpresera post 3rd presera post 3rd presera post 3rd 1 0 10,240 0 80 0 6400 80 0 1,280 0 80 2 0 20,480 0 320 0 640 0 160 0 640 0 5,120 4 0 5,120 040 0 5,120 0 160 0 10,240 0 640 a5 0 10,240 0 160 0 10,240 0 160 010,240 40 640 +control 49H.8 1:1.600 aTn 1:25,600 FACS MCF7 LSC  Mouse#12452⁰Ab alone IgGpresera10.02%10.49% 9.78% 9.78%  post3rd95.76%95.30%94.71%95.48% 1.28% IgMpresera11.52%10.48%11.36% 9.86% post 3rd89.79%95.61%95.96%94.49% 1.15%

IgGpresera10.11% 9.60% 9.93%10.59%  post3rd42.28%28.88%27.09%23.46% 1.18% IgMpresera 9.63%10.75%11.12%10.16% post 3rd58.23%93.47%96.28%93.23% 1.14%

Second Series of Experiments

Polyvalent (Heptavalent) Conjugate Vaccine for Prostate, Breast, Ovarianand Small Cell Lung Cancer

Tumor-specific antigens have been identified and pursued as targets forvaccines. The inventors' previous work has shown that monovalentvaccines utilizing the tumor antigens Globo H, Lewis^(y), GM2,glycosylated MUC-1, Tn(c), sTn(c), or TF(c) conjugated to KLH to be safewith local erythema and edema but minimal systemic toxicities. As aresult of vaccination with these monovalent vaccines, most patientsgenerated specific high titer IgM or IgG antibodies against therespective antigen-KLH conjugates. The present invention provides aheptavalent vaccine wherein the components of the monovalent vaccinesare combined and administered with an adjuvant as treatment forprostate, breast, ovarian and small cell lung cancer.

A vaccine consisting of a unique combination of seven tumor antigensadministered with a saponin immunological adjuvant QS-21 or GPI-0100.The antigens are glycosylated MUC-1-G5, Globo H, GM2, Le^(y), Tn(c),sTn(c), and TF(c). In each case the antigen is conjugated to KeyholeLimpet Hemocyanin (KLH).

The preferred ranges of the antigen and adjuvant doses are as follows:

-   -   Glycosylated MUC-1-G5: 0.1 to 30 μg;    -   Globo H: 0.1 to 100 μg;    -   GM2: 0.1 to 100 μg;    -   Le^(y): 0.1 to 60 μg;    -   Tn(c): 0.1 to 100 μg;    -   sTn(c): 0.1 to 100 μg;    -   TF(c): 0.1 to 30 μg;    -   QS-21: 25-200 μg;    -   GPI-0100: 1-25 mg.

Example 1

Phase I Clinical Trial Protocol Using the Heptavalent Vaccine

Example 2

Heptavalent Vaccine Immunogenicity Trial in Mice

1. Methods

2. Results

Example 1 Pilot Phase I Trial in Patients with Epithelial Ovarian,Fallopian Tube, or Peritoneal Cancer with a Polyvalent Vaccine-KLHConjugate+QS-21

-   1.0 PROTOCOL SUMMARY-   2.0 OBJECTIVE-   3.0 BACKGROUND AND RATIONALE-   4.0 VACCINE PREPARATION-   5.0 TREATMENT SCHEDULE AND DOSE-   6.0 EVALUATION DURING STUDY-   7.0 BIOSTATISTICAL CONSIDERATIONS (Endpoints)-   8.0 BIBLIOGRAPHY    1.0 Protocol Summary and Program Plan

Patients with epithelial ovarian, fallopian tube, or peritoneal cancerwho receive surgical cytoreduction and platinum/taxane containingchemotherapy have a significant chance of entering complete clinicalremission but unfortunately approximately 70% will eventually relapse.These patients in clinical remission have minimal residual disease, andare excellent candidates in which to evaluate novel consolidationstrategies in an attempt to improve outcome. This pilot polyvalentprotocol represents the culmination of a series of monovalent phase Ivaccine trials at the center demonstrating the immunogenicity of thevarious component antigens. It represents the transition between thephase I monovalent trial program in second remission, to the planneddevelopment of larger trials designed to evaluate efficacy. Immunizationwith the individual antigens selected for this vaccine has beenconsistently immunogenic in the majority of patients. No confirmedsystemic toxicity has occurred related to vaccine administration. It isexpected that the immunogenicity will remain unchanged, and that nosystemic toxicity will occur with polyvalent vaccine administration.Eligible patients for this pilot trial are those patients initially withstage II-IV disease in complete clinical remission following primarytherapy, or following relapse and re-induction to remission withadditional chemotherapy. In this trial, patients will receive an antigendefined vaccine with the following ganglioside components: a) GM2, b)Globo-H; the blood group related antigens: c) TF(c), d) s-Tn (c), e) Tn(c) f) Lewis-Y; and g) the protein antigen MUC-1-G5 (glycosylated). Theprimary endpoints of this pilot study are safety, and confirmation ofcontinued immunogenicity. The secondary endpoint will be to characterizethe nature and duration of the antibody response.

2.0 Objectives

2.1 The primary endpoints of this pilot study are to determine thesafety of polyvalent vaccine administration, and continuedimmunogenicity in patients prior to conducting a large, randomizedstudy.

2.2 The secondary endpoint is to further characterize the nature andduration of the antibody response generated by the polyvalent vaccine(ELISA and FACS)

3.0 Background and Rationale

3.1 Disease Background and Suitability for Treatment

In 1999, approximately 22,500 new cases of ovarian cancer werediagnosed, and it is estimated that 14,500 women died of the disease.Seventy-five percent of patients with ovarian cancer will have spreadbeyond the ovary at diagnosis. Standard primary treatment consists ofcytoreductive surgery followed by a platinum and paclitaxel containingchemotherapy regimen.¹ Many patients have no clinically measurabledisease at the end of primary treatment. A review of second-looklaparotomy, however, indicates that less than 50% of patients areactually free of disease.² Furthermore, nearly half of patients with anegative second look procedure are destined to relapse and requireadditional treatment.^(3,4) Overall, only approximately 30% of patientsremain disease free with currently available treatment. Given theminimal disease burden at the completion of primary therapy, thesepatients are ideal candidates in which to evaluate immune modulatingstrategies.

3.2 Rationale for Polyvalent Vaccines Designed Primarily for AntibodyProduction

Varied data exists in solid tumors to support the development of immunedirected therapy. Studies have emerged in patients with melanoma whichdemonstrate that naturally acquired^(5,6), or actively induced^(7,8)antibodies may improve outcome. In a large clinical trial reported byReithmuller et al., 189 patients with resected Dukes C colon carcinomawere randomized to receive observation versus postoperative treatmentwith murine antibody C017-1A that recognizes the KSA antigen. Toxiceffects were limited to infrequent constitutional symptoms. At medianfollow-up of five years, the death rate was 36% in the treated groupversus 51% in the observed group. The advantage of treatment wasdemonstrated in univariate (p=0.051) and multivariate (p=0.043) analysiswhen controlling for other known prognostic factors.⁹

The basis for cancer vaccines designed primarily for antibody inductionare the many preclinical models demonstrating the ability of passivelyadministered or actively induced antibodies to prevent tumor recurrence¹⁰, the increasing number of clinical trials where passivelyadministered monoclonal antibodies have demonstrated clinical efficacy,and the correlation of antibodies, naturally acquired or vaccineinduced, with improved prognosis in several different clinicalsettings.⁶

EL4 lymphoma naturally expresses GD2 ganglioside, which is recognized bymonoclonal antibody 3F8. Vaccines containing GD2 covalently conjugatedto KLH and mixed with immunological adjuvant QS21 are the optimalapproach to vaccination against GD2. Relatively higher levels ofantibody administered two or four days after intravenous tumor challengeor moderate titers induced by vaccine that were present by day two orfour after tumor challenge were able to eradicate disease in most mice.If antibody administration was deferred until day seven or ten, littleor no benefit could be demonstrated. If the number of cells in the EL4challenge was decreased, giving a longer window of opportunity, thevaccinations could be initiated after tumor challenge and goodprotection seen.⁷ These results are consistent with the need to initiateimmunization with vaccines inducing antibodies in the adjuvant setting,when the targets are circulating tumor cells and micrometastases.Patients with ovarian cancer in first remission meet these criteria, andunfortunately have a high “event rate” (ie. 80% will relapse) allowingfor the rapid assessment of the efficacy of this approach.

The basis for the inventors' emphasis on polyvalent vaccines is tumorcell heterogeneity, heterogeneity of the human immune response and thecorrelation between overall antibody titer against tumor cells andeffector mechanisms such as complement mediated cytotoxicity (CDC) orantibody dependent cell mediated cytotoxicity (ADCC). For example, usinga series of 14 tumor cell lines and monoclonal antibodies (mAbs) against3 gangliosides, investigators at MSKCC have shown that significant cellsurface reactivity analyzed by flow cytometry and CDC increased from 2-8of the cell lines using one of three mAbs to 13-14 of the cell lineswhen the 3 mAbs were pooled. The median CDC increased 4 fold with thepool of mAbs compared to the best single mAbs.¹¹ Cancers of the ovaryexpress a rich array of cell surface antigens making them especiallysuitable targets for polyvalent vaccines.

Cell surface antigens (especially carbohydrate cell surface antigens)have proven to be unexpectedly potent targets for immune recognition andattack of human cancers. Many of the more tumor-restricted monoclonalantibodies derived by immunization of mice with human tumor cells havebeen found to be directed against carbohydrate antigens expressed at thecell surface¹² Immunization against carbohydrate antigens resultsgenerally in an antibody response (see references for dissenting views),which is primarily IgM.¹³⁻¹⁵ These antibodies are known to induce CDC,inflammation, and phagocytosis of tumor cells by thereticulo-endothelial system (opsonization).¹⁶ Immunization against cellsurface protein antigens can induce a variety of B and T lymphocyteresponses. The T lymphocyte responses are difficult to quantify in thecontext of vaccination trials and are not the focus of this proposal.The antibody responses against protein antigens contain IgM and IgG,both of which can induce complement activation (with regard to IgGdepending on the subclass, IgG1 and IgG3 being optimal). IgG antibodiesof these subclasses can also induce ADCC.

Antibodies are the primary mechanism for active elimination ofcirculating pathogens from the bloodstream. They are ideally suited foreradication of free tumor cells and systemic or intraperitonealmicrometastases and they have accomplished this as described above in avariety of preclinical mouse experiments (reviewed inreferences).^(10,7) In adjuvant immunization trials, the primary targetsare individual tumor cells or early micrometastases which may persistfor long periods after apparent resection of all residual tumor.¹⁷ Aftersurgery and completion of chemotherapy is the ideal time for immuneintervention, and in particular for administration of cancer vaccinesaimed at instructing the immune system to identify and kill the fewremaining cancer cells. If antibodies of sufficient titer can be inducedagainst tumor antigens to eliminate tumor cells from the blood andlymphatic systems, aggressive local therapies, including surgery,radiation therapy and intralesional treatments might result in long termcontrol of even metastatic cancers.

3.3 Preliminary Studies for the Antigens

GM2 vaccines: Investigators at the center have been refining the abilityto induce antibodies against GM2 in melanoma patients for fifteen years,since first demonstrating that vaccines containing purified GM2 could bemore immunogenic than vaccines containing tumor cells expressing GM2.¹⁸Initially GM2 adherent to BCG was selected as optimal, inducing IgMantibodies in 85% of patients. This was the basis for a randomized trialcomparing immunization with BCG to immunization with GM2/BCG in 122patients with AJCC Stage 3 melanoma.⁸ The IgM antibodies had a mediantiter of 1/160 and were short lived (8-12 weeks). IgG antibody inductionwas rare. Antibody titers have been maintained for over three years byadministration of repeated booster immunizations at 3-4 month intervals.When comparing patients as randomized in this trial, no statisticallysignificant difference on overall or disease free survival was seen.Pre-existing GM2 antibodies were seen in 5 patients in the controlgroup, as opposed to one in the GM2 treated group which may have bluntedthe treatment result. The association between better outcome and thepresence of GM2 antibodies was seen (8).

TF, Tn and sTn vaccines: Patients with various epithelial cancers havebeen immunized with unclustered TF-KLH and sTn-KLH conjugate vaccinesplus various adjuvants.¹⁹ High titer IgM and IgG antibodies against TFand sTn antigens have resulted, but we found that the majority of thereactivity detected in sera from immunized mice and patients was againstantigenic epitopes present on synthetic constructs which were notpresent on naturally expressed mucins.²⁰ Based on previous studies withTn antigen,²¹ Kurosaka and Nakada et al. hypothesized that MLS102, amonoclonal antibody against sTn, might preferentially recognize clusters(C) of sTn. In studies at MSKCC with monoclonal antibody B72.3 and withsera raised against TF-KLH and sTn-KLH conjugate vaccines in mice and inpatients resulted in the same conclusion.^(20,22) The availability ofsynthetic TF, Tn and sTn clusters consisting of 3 epitopes covalentlylinked to 3 consecutive serines or threonines has permittedinvestigators at MSKCC to prove this hypothesis. In both direct testsand inhibition assays, B72.3 recognized sTn clusters exclusively, andsera from mice immunized with sTn (C)-KLH reacted strongly with bothnatural mucins and tumor cells expressing sTn.²² Based on thisbackground, we initiated trials with the TF(C)-KLH, Tn(C)-KLH andsTn(C)-KLH conjugate vaccines in patients with breast cancer. Antibodiesof relevant high titer specificity, including against OSM or PSM andcancer cells expressing TF, Tn or sTn, have been induced for the firsttime in the inventors' experience. Based on these results confirming theimportance of clustered epitopes and defining their relevantimmunogenicity, we are including these clustered antigens in thepolyvalent vaccine against ovarian cancer.

Le^(y) and Globo H vaccines: The development of Le^(y) and Globo Hvaccines was previously limited by the lack of sufficient quantities ofantigen for vaccine construction and testing. Over the last four years,Dr. Danishefsky has successfully synthesized both antigens.^(23,24)Investigators at MSKCC have immunized groups of mice with GloboH-ceramide plus or minus adjuvants QS-21 and Salmonella minnesota mutantR595, and with Globo H covalently attached to KLH or BSA plusimmunological adjuvant QS-21. The highest antibody titers against bothsynthetic antigen and MCF7 cells expressing Globo H were induced by theGlobo H-KLH plus QS-21 vaccine.^(23,24) The antibody titer inducedagainst synthetic Globo H was 1/120,000 by ELISA, the titer inducedagainst MCF7 was 1/320, and potent complement mediated cytotoxicity wasseen as well. Le^(y)-BSA and Le^(y)-KLH vaccines have also been testedin the mouse. High titer antibody responses have resulted against thesynthetic epitope of Le^(y) and against tumor cells expressing Le^(y) inthe majority of mice immunized. Based on these results, monovalent phaseI clinical trials with Globo H-KLH plus QS-21 and Le^(y)-KLH plus QS21have been initiated in patients with breast, prostate or ovary cancer.Antibodies against the purified antigens and against tumor cellsexpressing these antigens were induced in most patients and themanuscript was recently published for the latter.^(26,27)

MUC1 and MUC2 vaccines: Investigators at MSKCC have immunized mice withMUC1-KLH and MUC2-KLH, plus QS-21, and seen induction of consistent hightiter IgM and IgG antibodies against MUC1 and MUC2 and human cell linesexpressing MUC1 and MUC2, as well as protection from a syngeneic mousebreast cancer expressing human MUC1 as a consequence of genetransduction. Mice were also immunized with vaccines containing MUC1peptides of various lengths conjugated to KLH by one of three methods ornot, and mixed with QS-21 or BCG. MUC1 containing 30 amino acids ormore, conjugated to KLH with an MBS bifunctional linker and mixed withimmunological adjuvant QS-21 induced the highest titer antibodies.²⁸Based on these studies in the mouse, a trial was initiated and completeda trial with this MUC1-KLH plus QS-21 vaccine in breast cancer patientswho were free of detectable breast cancer after resection of all knowndisease. Nine patients were treated with a 31 amino acid MUC1 peptidewith cysteine at one end for conjugation to KLH and the immune dominantepitope-APDTRPA at the other end.²⁹ No patient had detectable MUC1serological reactivity by ELISA or FACS prior to immunization. Theresults are summarized below in table below. Reactivity against MUC1 andtumor cells expressing MUC1 was seen in most patients. A separate groupof patients were immunized with MUC2-KLH plus QS21. Analysis of thistrial is not yet complete, but the results to date are also summarizedin the table below.

The inventors have been unable to demonstrate T-lymphocyteproliferation, interferon γ and IL4 release by ELISPOT assays, CTLactivity or positive DTH responses after vaccination with MUC1 or MUC2.The proliferation assays were particularly focussed on in the MUC1trials. Patients had leukophoresis pre and post vaccination, providingample lymphocytes for study. After 2 years of steady endeavor, there hasbeen no clear evidence of augmented reactivity against MUC1 peptides ofvarious lengths or, in HLA A2 positive patients against heteroclyticMUC1 peptides with single amino acid changes that increased binding toHLA A2. (personal communication, P. O. Livingston) Pre and postvaccination PBLs from the first 6 patients vaccinated with MUC1 werealso sent to the laboratory of Dr. Olivera Finn for CTL precursorfrequency analysis. No increase in frequency was seen. Over all, themajor difference between results from MSKCC with the 31aa MUC1-KLH plusQS-21 vaccine and Dr. Finn's results with a 104aa MUC1 peptide plus BCGvaccine was that the former had a clearly demonstrable, consistentantibody response, which was reactive with tumor cells. Inhibitionassays were performed to better understand this serologic response.³⁰Much of the IgM response and nearly all of the IgG response were againstthe immune dominant epitope, APDTRPA, preferentially with RPA at theterminal position.

KSA vaccines: KSA has been prepared in the baculovirus system by JennerTechnologies (San Ramon, Calif.) and 10 mcg/patient has been providedfor testing. Due to the small quantity of KSA available, followingdemonstration of relevant immunogenicity in mice, we treated groups of 9patients with KSA plus QS21 or with KSA covalently linked to KLH byglutaraldehyde, plus QS21. In neither case was there significantinduction of antibodies against KSA that had not been glutaraldehydetreated, or tumor cells expressing KSA. Consequently KSA will not beincluded in the polyvalent vaccine. The results of this and the othertrials with KLH conjugate vaccines are summarized in the table below(personal data, P. O. Livingston).

Summary of Serological Results in Vaccinated Patients Median MedianELISA IgG FACS Median Median Antigen IgM IgG Subclass IgM IgG IA CDC GM2640 320 IgG1+3 +++ ++ ++ ++ Globo H 640 40 IgG1+3 ++ + ++ + Lewis^(y) 800 ++ + + + Tn 1280 1280 ++ − + − STn 1280 160 IgG3 +++ − + − TF 320 10− + − MUC1 1280 5120 IgG1+3 + − + − MUC2 2560 2560 pending KSA 40 160 −− − −

Additional variables: Two additional variables have proven critical, themethod of conjugation and the epitope ratio of antigen molecules per KLHmolecule. The optimal conjugation approached has varied with theantigen. Gangliosides are best conjugated using ozone cleavage of theceramide double bond and introducing an aldehyde group followed bycoupling to aminolysyl groups of KLH by reductive amination. Thisapproach was not as effective for conjugation of Tn, sTn and TF clustersor Globo H to KLH where an M2C2H linker arm has proved most efficient³¹or for MUC1 or MUC2 where an MBS linker group was optimal.²⁸

The impact of dose and schedule of vaccine administration on antibodyresponse to GM2 vaccines in melanoma patients has also been explored.Immunization 4 times at weekly intervals or biweekly intervals followedby booster immunizations twice at 2-3 month intervals was compared to 6immunizations at monthly intervals. Initial immunizations at weekly orbiweekly intervals resulted in comparable high titers (with high titersoccurring slightly sooner at weekly intervals), but remarkably themonthly immunizations resulted in far weaker or undetectable antibodyresponses in the 6 patients vaccinated.³² GM2-KLH plus QS-21 vaccinesprepared at MSKCC and at Progenics Pharmaceuticals have each been testedin dose finding studies such as those proposed in this application. Inboth cases GM2 doses of 3 ug or less resulted in lower IgM titers andundetectable IgG titers in most patients. GM2 doses of 10, 30 and 100 uggave comparable IgM and IgG titers.³³ Based an these studies, we haveselected the initial weekly schedule of 3-4 immunizations followed bybooster immunizations every three months, and the doses for use in therandomized Phase III trial.

3.4 Potential Toxicity of Vaccination

The expected safety of the vaccine is based on the safety of vaccinationwith the individual components. Clinical experience is growing inclinical trials with vaccine induced antibody responses against each ofthe included antigens. Antigen expression at secretory borders in thesetrials, where the majority is located, has induced neither immunologicaltolerance nor symptomatic autoimmunity once antibodies are present,suggesting they are sequestered from the immune system. Nevertheless, aregular schedule of laboratory studies and physical examinations aredesigned to detect any abnormalities. This pilot trial will representone of the first studies to confirm the safety of polyvalent vaccineadministration in this setting.

3.5 General Immune Approaches

Various methods have been used to increase the immunogenicity ofantigens, and in particular for inducing an IgG response. In preclinicallaboratory studies, we have found the covalent attachment of antigen tokeyhole limpet hemocyanin (KLH) to be most effective.³⁴ KLH is welltolerated, and has previously caused only mild inflammation at thevaccine injection site. Attachment of KLH may be accomplished by avariety of cross-linking methods. MBS(m-maleimidobenzoly-N-hydroxysuccinimide ester) is the best-knownheterobifunctional reagent; and at neutral pH cross-links thiol groupswith amino groups. The linkage proceeds via two separate reactions, thuslimiting bonds between identical molecules. In addition to linkingantigen to immunogenic carrier proteins, the titer of antibody inducedmay be further augmented with the use of appropriate immunologicaladjuvants. We have immunized groups of melanoma patients with vaccineseither containing no adjuvant, or using DETOX, BCG or QS-21. QS-21 is asignificantly more effective adjuvant than the others, producingsignificantly higher titer IgM and IgG antibodies. It is a saponinderivative extracted from the bark of the South American tree Quillajasaponaria Molina. The monosaccharide composition, molecular weight,adjuvant effect and toxicity for a series of these saponins have beendescribed.^(35,36) It has also proven to be non-toxic and effective ataugmenting the immunogenicity of an FeLV subunit vaccine in cats 37 andan HIV-1 recombinant vaccine in Rhesus monkeys. A phase I trialdemonstrating the safety and suggesting the efficacy of a 100 ug QS-21dose in patients treated with GM2-KLH vaccines has been reported. Theonly adverse events reported were minimal flu-like symptoms, and milddiscomfort at the injection site.³⁸ Thus, conjugation with KLH and theaddition of QS-21 have become standard approaches for vaccineconstruction at MSKCC has proven optimal for antibody induction againsta variety of gangliosides, MUC1, MUC2, KSA, Tn, sTn, TF, Le^(y) andGlobo H in the mouse and in humans.

3.6 Distribution of the antigens studied: In general, the antigenscontained in this vaccine are expressed on ovarian cancer cells withhigh frequency. Recent studies at MSKCC have characterized thisdistribution in a variety of tumor types including ovarian cancer usingimmunohistochemical staining. A variety of tumor specimens were used ineach tumor type, and it was required that 50% or more cells be positivein order to consider the antigen present. The presence of these antigenson the tumor specimens tested in ovarian cancer was: GM2 (100%), GLOBO-H(60%), MUC 1 (100%), sTn (60%), TF (100%), Le Y (80%). ³⁹-⁴¹

3.7 Rational for the inventors' approach: The inventors' hypothesis isthat induction of an antibody response against several cell surfaceantigens on ovarian cancer cells with a polyvalent conjugate vaccinewill result in eradication of free tumor antigen, circulating tumorcells and micrometastases. The polyvalent nature of the vaccine andantibody response is important to eliminate escape by tumor cells thatfail to express any one or two of the antigens, and to increase thenumber of antibodies reacting against each cell. It is expected that theinventors' vaccine will prove consistently immunogenic against five orsix of the ovarian cancer cell surface antigens described above, andthat it will prove nontoxic. Whether immunization with this polyvalentvaccine in high-risk ovarian cancer patients in the remission settingwill result in prolonged disease-free and overall survival is the focusof subsequent studies to follow this pilot trial.

4.0 Vaccine Preparation

4.1 GM2 is provided by Progenics. GLOBO-H, Lewis-Y, TF(c), Tn (c), andsTn (c) are synthesized in the laboratory of Dr. Sam Danishefsky at thecenter. MUC-1 is produced at the core facility at MSKCC. Forglycosylation, the MUC-1 was shipped to the University of Copenhagen,Glycobiology Group. The glycosylation was carried out by GalNActransferase using UDP-N-GalNAc as substrate. The product was shippedback to MSKCC after purification by reverse phase HPLC.

4.2 QS-21 is obtained from Aquila Biopharmaceuticals in 100 mg vials asa white powder and is stored at −30 degrees Celsius. This is suspendedinitially in PBS as it is less soluble in normal saline and then finaldilutions are made in normal saline. QS-21 is passed through a 0.22micrometer filter immediately prior to use.

4.3 Conjugation to KLH is accomplished with three different conjugationprocedures: direct amination (ozonolysis) for GM2, the M2C2Hbifunctional linker group for Globo H and Le Y, and the MBS bifunctionallinker group for the four mucin antigens.

4.4 The sterility of the conjugate is confirmed by passage through a0.22 micrometer filter and it is stored in frozen normal saline at −30to −80 degrees Celsius.

4.5 Vials are released for use following standard lot release testing(approximately five weeks).

5.0 Treatment Schedule and Dose

5.1 Vaccine Contains:

[GM2(10 mcg)/TF(c)(3 mcg)/sTn(c) (3 mcg)/Globo-H(10 mcg)/MUC1-1-G5(3mcg)/Le^(Y) (10 mcg)/Tn(c) (3 mcg)]-KLH (≈400 mcg) with adjuvantQS21(100 mcg)

5.2 Immunization Schedule

The vaccine will be administered at weekly intervals for 3 doses. Thiswill be followed by a four week break and then a fourth vaccination.There will then be an eight week break and then a fifth vaccination,followed by additional immunizations every twelve weeks for 24 monthstotal (as long as patient remains on study).

IMMUNIZATION SCHEDULE WEEK # 1 2 3 7 15 VACCINE # 1 2 3 4 5

5.3 Dose Administration and Modification

5.3.1 No dose escalation or dose modification will be performed.Systemic toxicity has not been seen with the previous vaccine studies.Systemic toxicity >grade II (with the exception of fever withoutinfection) thought related to vaccination would result in removal of thepatient from study and suspension of the protocol pending investigation.

5.3.2 The preparation will be administered subcutaneously to a site inthe shoulders, buttocks, or thighs. It will be administered in onesyringe, and will be supplied in approximately 1 cc total volume.

6.0 Evaluation During Study

6.1 Immune Function (Summarized in Table)

6.1.1 Antibody response: Peripheral blood (20-30 ml) will be drawnaccording to the schedule in table 8.3 with the exception of week 0, 7,and 9 at which time 50-60 ml will be collected for antibody testing.Thereafter, blood will be drawn at 12-week intervals as the patientsreturn for booster immunizations. Antibodies against various antigenswill be studies by ELISA, and against human tumor cell lines by FACSwhen appropriate.

6.2 Clinical and Laboratory Assessment:

6.2.1 Clinical and laboratory assessment schedule: The clinical andlaboratory assessment schedule is outlined in Section 8.3 and includesparameters to assess the safety of the vaccine, as well as evaluate forsigns of disease recurrence and progression. Abnormal findings will beevaluated per standard medical practice, and the abnormality will beclassified as related to treatment, disease progression, or neither.

6.2.3 Extent of disease evaluation: All patients are by definitionwithout clinical or radiographic evidence of disease at protocol entry.

6.2.4 Radiographic imaging (CT abdomen and pelvis) will be obtained q 6months while on study, or if any clinical symptoms/examination findingswarrant further evaluation, or if serum CA-125 rises to >70 (per time totreatment failure criteria), confirmed by repeat value; or at any timeat the discretion of the investigator.

6.2.5 Length of follow-up: The primary endpoint of the study in thispilot trial is safety. Patients will be followed for the duration of thestudy, but based on previous trials, antibodies are generally present bythe seventh week, and we will proceed with the proposal for additionalstudies to evaluate efficacy if no systemic toxicity is seen in anypatient at the ninth week assessment. An additional 8-12 weeks would berequired for processing before patients could be enrolled on thepolyvalent study, allowing ample time for follow-up of the pilot trial.Patients will be followed until time to treatment failure, or until allvaccinations are completed (maximum 24 months).

6.3 Summary of Evaluation:

SUMMARY OF CLINICAL, LABORATORY, AND IMMUNE ASSESSMENTS week # 0^(a) 1 23 5 7 9 13 15 17 27^(c) vaccine # 1 2 3 4 5 V OfficeVisit * * * * * * * * * * * Hx and PE * * * * * * * * *CA-125 * * * * * * CBC, diff * * * * * * * * * * Hepaticprofile + * * * * * * * * * * creatinine Amylase * * * * * * * * * *Urinalysis * * * * * PT * Stool guaiac^(b) * * * * TSH * *Immune^(d) * * * * * * * bloods ^(a)= pretreatment evaluation, within 3weeks ^(b)= stool guaiac may be obtained by digital exam or cardscollected by patient ^(c)= following week 27, visits + laboratorystudies + immunization q 3 months, CT scan q 6 months while on study^(d)= immune bloods routinely consist of 20-30 ml collected in 3 red toptubes. In order to obtain sufficient serum to evaluate for multipleantibodies, 6 tubes will be collected instead of 3 at thepre-vaccination visit; and at week 7 and 9 only.7.0 Biostatistical ConsiderationsEndpoints:

The primary endpoints of this pilot trial are safety and confirmation ofimmunogenicity in the polyvalent setting. No systemic toxicity hasoccurred with the administration of monovalent vaccines at the center.Toxicity is not expected with this preparation. Following this pilot,additional studies with efficacy as the endpoint will be proposed. Thispilot trial would be suspended pending investigation for any systemictoxicity thought related to vaccine in any patient. The same criteriafor immunogenicity will be used as that of the individual pilot trials:patients must have IgM titer >1:80, or a four fold increase inprevailing antibody titer if present at baseline. Nine patients will beaccrued, and >5 of 9 patients should meet these criteria for three ormore antigens in order to proceed with this construct in additionalstudies. In prior trials, antibodies are generally present by completionof the fourth vaccination (week 7). If no systemic toxicity is seen bythe week 9 assessment in these patients, we will proceed with proposalsfor phase II studies with efficacy endpoints.

While not the endpoint of this pilot, patients would be removed fromstudy for relapse as defined below. Time to treatment failure will besimply defined based on data from Rustin et al.⁴² Treatment failure canbe characterized by 1) physical examination evidence of diseaserecurrence, radiographic evidence of disease recurrence (biopsy will beperformed at the discretion of the principal investigator but is notrequired); or 3) CA-125 elevation to twice the upper limits of normal(ie. ≧70), confirmed by a second sample also ≧70 U/ml. Time to treatmentfailure for biochemical relapse is recorded as the date of the firstsample ≧70 U/ml.

The secondary endpoint of this pilot trial is to characterize the natureand duration of the immune response. Peripheral blood (20-30 ml) will bedrawn as indicated in the table. Antibodies against the individualantigens will be studies by ELISA, and against the appropriate humantumor antigen FACS using human tumor cell lines expressing therespective antigen.

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Example 2 Heptavalent Vaccine Immunogenicity Trial in Mice Methods

Serological Analyses

1. ELISA (Enzyme-Linked Immunosorbent Assay):

ELISA assays were performed as described below. Antigen in ethanol or in0.1 M carbonate buffer (pH 11) were coated on ELISA plates at 0.2μg/well for glycolipids and 0.1 μg/well for peptides. Serially dilutedantiserum was added to each well and alkaline phosphatase-conjugatedgoat anti-mouse IgM or anti-mouse IgG was added at a dilution of 1:200(Southern Biotechnology Associates, Inc, Birmingham, Ala.). Goatanti-mouse IgG and IgM conjugated with alkaline phosphatase obtainedfrom Kierkegaard and Perry Labs, (Gaithersburg, Md.) were used as secondantibodies. ELISA titer is defined as the highest dilution yielding anabsorbance of 0.1 or greater over that of normal control mouse sera.

2. Cell Surface Reactivity Determined by FACS:

The cell surface reactivity of immune sera was tested on human MCF-7 andLSC cell lines. Single cell suspensions of 2×10⁵ cells/tube were washedin PBS with 3% fetal calf serum (FCS) and 0.01M NaN₃ and incubated with20 μl of 1:20 diluted sera or monoclonal antibody mAb for 30 min on ice.After two washes with 3% FCS in PBS, 20 μl of 1:15 diluted goatanti-mouse IgM or IgG-labeled with fluorescein-isothiocyanate (FITC,Southern Biotechnology Associates Inc. Birmingham, Ala.) was added, andthe mixture incubated for 30 min. After a final wash, the positivepopulation and mean fluorescence intensity of stained cells weredifferentiated using FACScan, Becton & Dickinson Immunocytometry, SanJose, Calif.

Immunization of mice with Heptavalent-KLH Conjugates plus

ELISA

ELISA plate mice coated immunized with . . . / pre vacc. post vacc. with. . . (Group#) (IgG/IgM) (IgG/IgM) Comments GM2 GM2 (group #1) 0/0 0/0GloboH (group #2) 0/0 LeY (group #3) 0/0 Muc1-G5 (group #4) 0/0 STn(c)(group #5) 0/0 TF(c) (group #6) 0/0 Tn(c) (group #7) 0/0 Globo H GM2(group #1) GloboH (group #2) 0/0  640/5120 LeY (group #3) Muc1-G5 (group#4) STn(c) (group #5) TF(c) (group #6) Tn(c) (group #7) LeY GM2 (group#1) 0/0 GloboH (group #2) 0/0 LeY (group #3) 0/0  640/2560 Muc1-G5(group #4) 0/0 STn(c) (group #5)  0/160 TF(c) (group #6)  0/40 Tn(c)(group #7)  0/40 Muc1-G5 GM2 (group #1)  0/320 GloboH (group #2)  0/160LeY (group #3)  0/160 Muc1-G5 (group #4) 0/0 2560+/1280   STn(c) (group#5) 40/80 TF(c) (group #6) 40/80 Tn(c) (group #7)  0/320 mAb. C595 2560/B55 /<5120+++ STn(c) GM2 (group #1) 0/0 GloboH (group #2) 0/0 LeY (group#3) 0/0 Muc1-G5 (group #4) 0/0 STn(c) (group #5) 0/0 5120/320  TF(c)(group #6) 0/0 Tn(c) (group #7) 0/0 mAb. cc49 1280/0   Tf(c) GM2 (group#1)  0/40 GloboH (group #2) 40/80 LeY (group #3) 40/40 Muc1-G5 (group#4) 5120+/160    STn(c) (group #5) 5120+/320    TF(c) (group #6) 0/05120+/320    Tn(c) (group #7) 5120+/640    mAb. JAA-F11 0/ A78-6/A7 /640Tn(c) GM2 (group #1) 0/0 GloboH (group #2) 0/0 LeY (group #3) 0/0Muc1-G5 (group #4) 5120+/160    STn(c) (group #5) 5120+/320    TF(c)(group #6) 5120+/320    Tn(c) (group #7) 0/0 5120+/2560   mAb. 5F4/5120++ HB-Tn1 /5120

ELISA

Immunization of mice with Heptavalent-KLH Conjugates* plus GPI-100 orQS-21. ELISA (IgG/IgM) FACS (IgG/IgM) Group # Antigen Mice # Pre-vacc.Post-vacc Pre-vacc. Post-vacc. Comments 8. Heptavalent-KLH + 100 ugTn(c) 1 0/0 5120+/160 GPI-100 2 0/0 5120/160 (200 ul/mouse) 3 0/05120/160 4 0/0 5120++/640 5 0/40 5120+/320 Tf(c) 1 0/0 5120/160 2 0/05120/160 3 0/0 5120/160 4 0/0 5120+/320 5 0/40 5120+/640 sTN(c) 1 0/05120/640 2 0/0 1280/640 3 0/0 5120/640 4 0/0 5120/320 5 0/0 1280/320MUC1-1G5 1 0/0 5120+/0 2 0/0 5120+/80 3 0/0 5120+++/160 4 0/0 5120++/405 0/0 5120+++/80 Ley 1 0/0 0/40 2 0/0 320/640 3 0/0 0/160 4 0/0 80/40 50/0 0/640 Globo H 1 0/80 40/640 2 0/80 0/640 3 0/80 0/640 4 0/16040/1280 5 0/160 0/2560 GM2 1 0/0 0/40 2 0/0 0/0 3 0/0 0/0 4 0/0 0/0 50/0 0/0 Apr. 2, 2001 9. Heptavalent-KLH + 100 ug Tn(c) 1 0/0 640/40GPI-100 2 0/0 640/0 (Batch J) 3 0/0 5120/40 (200 ul/mouse) 4 0/0 5120+/05 0/0 1280/40 Tf(c) 1 0/0 1280/80 2 0/0 1280/40 3 0/0 5120+/40 4 0/05120++/40 5 0/0 2560/40 sTN(c) 1 0/0 320/80 2 0/0 320/160 3 0/0 640/1604 0/0 640/80 5 0/0 80/320 MUC1-1G5 1 0/0 2560/0 2 0/0 640/0 3 0/0 640/04 0/0 640/0 5 0/0 2560/80 Ley 1 0/0 0/0 2 0/0 0/80 3 0/0 0/0 4 0/0 0/0 50/0 160/1280 Globo H 1 0/80 0/160 2 0/80 0/320 3 0/80 0/160 4 0/80 0/3205 0/80 40/320 GM2 1 0/0 0/0 2 0/0 0/0 3 0/0 0/0 4 0/0 0/0 5 0/0 0/0 10.Heptavalent-KLH + 100 ug Tn(c) 1 0/0 5120++/320 GPI-100 + polysorbate 20/0 5120/320 80 3 0/40 5120+/1280 (200 ul/mouse) 4 0/0 5120+/640 5 0/02560/320 Tf(c) 1 0/0 5120+/320 2 0/0 5120+/160 3 0/0 5120+/640 4 0/05120+/640 5 0/0 5120+/320 sTN(c) 1 0/0 320/640 2 0/0 320/1280 3 0/01280/1280 4 0/0 640/40 5 0/0 320/320 MUC1-1G5 1 0/0 1280/40 2 0/05120/80 3 0/0 5120/160 4 0/0 2560/160 5 0/0 5120/40 Ley 1 0/0 0//0 2 0/00/0 3 0/0 0/640 4 0/0 0/640 5 0/0 0/80 Globo H 1 0/40 80/640 2 0/8040/2560 3 0/320 0/2560 4 0/160 0/2560 5 0/80 0/640 GM2 1 0/0 0/0 2 0/00/0 3 0/0 0/0 4 0/0 0/0 5 0/0 0/0 11. Heptavalent-KLH+ + 10 ug Tn(c) 10/0 5120/160 QS-21 2 0/0 5120+/160 (200 ul/mouse) 3 0/40 5120+++/160 40/80 2560/320 5 0/0 5120+/80 Tf(c) 1 0/40 5120+/320 2 0/0 5120++/640 30/160 5120++640 4 0/320 2560/640 5 0/40 5120++/320 sTN(c) 1 0/0 40/40 20/0 2560/640 3 0/0 5120/160 4 0/0 1280/80 5 0/0 5120/640 MUC1-1G5 1 0/01280/160 2 0/0 2560/80 3 0/0 5120/80 4 0/0 2560/320 5 0/0 5120++/80 Ley1 0/0 0//0 2 0/0 0/0 3 0/0 0/0 4 0/0 0/0 5 0/0 40/40 Globo H 1 0/8040/320 2 0/80 80/320 3 0/80 40/640 4 40/160 0/640 5 0/320 0/2560 GM2 10/0 0/0 2 0/0 0/0 3 0/0 0/0 4 0/0 0/0 5 0/0 0/0 Apr. 16, 2001 12.Heptavalent-KLH + 3 ug Tn(c) 1 0/40 2560/160 ER-803022 2 0/0 640/160(200 ul/mouse) 3 0/40 320/320 4 0/0 160/160 5 0/0 2560/160 Tf(c) 1 0/02560/40 2 0/0 2560/40 3 0/0 640/160 4 0/0 320/80 5 0/0 2560/0 sTN(c) 10/0 0/40 2 0/0 40/80 3 40/40 80/160 4 0/0 160/160 5 0/0 40/0 MUC1-1G5 10/0 80/0 2 0/0 80/0 3 0/0 80/40 4 0/0 40/80 5 0/0 40/0 Ley 1 0/0 0/0 20/0 0/0 3 0/0 0/0 4 0/0 0/80 5 0/0 0/0 Globo H 1 0/80 0/320 2 0/16040/1280 3 0/320 0/320 4 80/160 160/1280 5 40/160 320/640 GM2 1 0/0 0/0 20/0 0/0 3 0/0 0/0 4 0/0 0/0 5 0/0 0/0 13. Heptavalent-KLH + 10 ug Tn(c)1 0/40 40/320 ER-803732 2 0/0 40/40 (200 ul/mouse) 3 0/0 320/160 4 0/40160/160 5 0/40 160/80 Tf(c) 1 0/0 160/160 2 0/0 320/40 3 0/40 640/160 40/40 320/160 5 0/0 320/160 sTN(c) 1 0/0 40/80 2 0/0 0/80 3 0/0 0/320 40/160 40/320 5 0/40 40/160 MUC1-1G5 1 0/0 640/40 2 0/0 0/40 3 0/0 40/804 0/40 80/160 5 0/0 160/80 Ley 1 0/0 0/0 2 0/0 0/0 3 0/40 40/320 4 0/00/40 5 0/0 0/80 Globo H 1 40/80 80/160 2 0/160 40/320 3 0/40 80/320 40/80 0/160 5 80/160 160/320 GM2 1 0/0 0/0 2 0/0 0/0 3 0/0 0/0 4 0/0 0/05 0/0 0/0 14. 30 ug KLH + 100 ug Tn(c) 1 0/0 0/160 GPI-100 2 0/0 0/40(100 ul/mouse) 3 0/0 0/0 4 0/0 0/160 5 0/40 0/640 Tf(c) 1 0/40 0/320 20/40 0/160 3 0/0 0/80 4 0/0 0/320 5 0/160 40/640 sTN(c) 1 0/0 0/80 2 0/00/40 3 0/0 0/0 4 0/0 0/320 5 0/0 0/80 MUC1-1G5 1 40/40 0/160 2 0/40 0/803 0/0 0/80 4 0/0 0/320 5 0/0 0/320 Ley 1 0/0 0/40 2 0/0 0/0 3 0/0 0/0 40/0 0/0 5 0/0 0/0 Globo H 1 0/320 40/1280 2 0/320 0/640 3 0/80 0/1280 40/160 0/640 5 0/160 40/640 GM2 1 0/0 0/160 2 0/0 0/40 3 0/0 0/0 4 0/00/320 5 0/0 0/160 KLH 1 0/0 5120++/320 2 0/40 5120++/160 3 0/05120++/320 4 0/0 5120++/640 5 0/40 5120++/320 Heptavalent: Tn(c) (3 ug),TF(c) (3 ug), sTn(c) (3 ug), MUC1-1G5 (3 ug), Ley (10 ug), Globo H (10ug), and GM2 (10 ug). New GPI-100 used for all groups except group 9(batch J). Polysorbate 80, 4 mg/ml in final vaccine.

Immunization of mice with Heptavalent-KLH Conjugates* plus GPI-100 orQS-21. FACS % positive cells Group # MCF-7 (IgG/IgM) LSC (IgG/IgM) (miceimmunized with . . . ) Mice # Pre-vacc. Post-vacc. 1:200 dilut.Pre-vacc. Post-vacc. 1:200 dilut. 1. 3 ug GM2-KLH + 100 ug 1 11.79/10.6821.45/11.79 15.73/0.38 10.90/9.58 6.91/2.93 6.56/0.14 GPI-100 210.23/10.69 4.80/1.49 2.34/0.0 10.90/10.45 5.99/3.45 1.45/0.59 (100ul/mice) 3 9.50/10.74 6.95/15.79 0.99/0.21 11.29/11.11 7.79/1.536.41/0.23 4 9.81/9.68 3.41/12.63 0.99/2.06 9.76/11.06 6.94/1.753.42/0.02 5 10.81/10.42 10.43/3.15 12.29/0.11 10.13/10.76 4.40/1.670.40/0.10 2. 3 ug GloboH-KLH + 100 ug 1 10.63/9.93 5.79/17.26 6.60/0.199.87/10.99 7.08/2.77 9.55/0.25 GPI100 2 10.08/10.67 16.60/27.6310.54/6.75 10.0/10.41 8.9/3.43 1.29/0.52 (100 ul/mice) 3 11.03/10.0927.21/40.86 27.58/2.30 10.42/10.61 11.56/1.15 7.60/0.02 4 10.16/11.361.92/13.77 2.30/1.03 10.57/11.04 5.29/2.43 3.81/0.24 5 11.94/10.8224.61/12.97 20.32/3.62 10.79/10.72 10.58/2.54 1.25/0.20 3. 3 ugLey-KLH + 100 ug 1 11.01/9.97 85.01/43.57 41.26/1.73 10.22/10.1089.17/44.27 13.40/1.87 GPI-100 2 9.95/9.69 47.75/78.17 4.86/3.4310.49/8.39 91.69/79.9 7.64/7.04 (100 ul/mice) 3 10.41/10.58 69.55/10.9520.53/0.12 11.30/9.47 95.57/21.25 24.67/0.16 4 11.93/10.03 91.08/0.7621.29/0.0 10.96/9.60 81.5/6.24 9.08/0.38 5 12.46/11.18 42.94/36.6713.66/1.87 10.98/10.61 12.74/52.16 1.05/1.87 4. 3 ug Muc-1G-5-KLH + 100ug 1 11.33/10.94 98.12/9.52 97.98/0.93 9.50/9.89 4.83/1.10 2.29/0.18GPI-100 2 10.90/9.44 88.05/1.47 66.29/0.07 11.39/9.63 8.47/0.15 4.85/0(100 ul/mice) 3 10.59/10.81 93.34/0.93 83.12/0.09 10.42/9.85 7.42/1.163.15/0.02 4 10.47/9.70 94.57/21.92 85.52/0.47 9.70/11.11 10.99/7.793.47/0.71 5 12.29/10.03 99.77/6.12 99.79/0.77 *11.05/10.49 63.71/1.8228.90/0.71 5. 3 ug STn(c)-KLH + 100 ug 1 9.56/9.77 13.95/1.74 1.29/0.1310.10/10.73 95.14/4.42 86.94/0.50 GPI-100 2 9.91/10.59 15.95/1.7912.70/0.23 10.20/11.18 98.65/19.42 88.58/0.59 (100 ul/mice) 310.72/10.55 6.88/2.81 6.23/0.02 11.31/11.46 97.78/31.98 95.31/0.36 49.62/9.52 4.62/1.69 3.18/0.14 10.11/9.78 94.14/1.19 86.65/0.09 510.77/10.71 17.96/2.16 7.17/0.3 10.71/0.05 97.66/7.28 87.09/0.38 6. 3 ugTf(c)-KLH + 100 ug 1 10.84/10.39 13.89/5.73 5.86/0.23 9.64/10.317.66/4.74 3.85/0.24 GPI-100 2 9.22/11.34 31.03/4.44 31.75/0.109.65/10.15 9.01/1.17 3.98/0.18 (100 ul/mice) 3 10.59/10.11 4.05/1.050.73/0.02 10.12/9.92 4.97/2.77 0.90/0.04 4 10.26/10.63 6.72/7.102.19/0.19 9.72/10.79 3.85/3.58 2.25/0.16 5 12.0/10.90 9.11/6.285.18/0.18 *11.54/10.09 11.71/5.0 12.76/0.54 7. 3 ug Tn(c)-KLH + 100 ug 19.60/10.44 3.64/8.41 0.62/0.37 10.65/10.46 9.91/5.07 3.02/0.32 GPI-100 210.54/10.46 12.94/3.24 20.35/0.17 11.50/10.85 4.81/0.48 5.33/0.05 (100ul/mice) 3 10.51/10.46 4.72/23.22 0.16/1.22 10.70/9.68 2.37/1.330.04/0.02 4 10.97/9.48 8.50/17.77 4.52/0.16 10.94/10.97 18.50/1.814.18/0.12 5 10.79/10.72 46.27/32.77 9.46/2.04 11.40/10.16 25.42/16.8416.5/22.80 *DU145 + control IE-3 91.67, 95.15 + control IE-3 95.73%MLS128 77.07% HMFG1 38.34% MLS132 19.92, 4.52 49H8 84.90% 8.Heptavalent-KLH + 100 ug 1 10.84/11.21 89.44/17.63 69.48/0.4710.29/10.63 97.79/49.78 90.80/0.46 GPI-100 2 9.55/9.54 96.98/25.8783.67/1.12 9.97/10.58 95.44/74.27 83.11/0.35 (200 ul/mice) 3 9.94/10.8896.53/32.44 88.36/0.79 10.56/10.91 95.39/40.29 85.79/0.49 4 10.90/10.7299.47/5.72 96.61/0.45 10.69/10.42 97.65/16.44 88.77/0.28 5 10.37/10.1775.64/10.67 63.57/0.11 10.58/10.14 93.93/15.49 71.06/1.36 9.Heptavalent-KLH + 100 ug 1 10.65/10.57 74.25/12.81 69.56/1.0710.93/10.21 99.45/8.83 85.59/0.38 GPI-100 2 10.20/10.97 98.22/16.5181.61/0.91 10.86/10.78 96.70/22.94 66.20/0.50 (batch J) 3 9.96/9.6889.36/1.03 60.88/0.22 9.79/9.80 95.10/0.43 53.45/0.03 (200 ul/mice) 410.77/10.75 82.25/8.95 27.80/0.09 10.58/10.28 92.21/1.17 65.13/0.18 510.95/10.77 77.49/10.19 62.39/0.13 10. Heptavalent-KLH + 100 ug 110.99/10.32 84.54/8.61 39.91/0.49 10.63/10.92 93.11/38.33 63.61/1.57GPI-100 + polysorbate 2 10.09/10.92 97.83/11.79 94.21/0.16 10.72/10.6897.64/12.52 77.95/0.05 80 3 11.32/10.32 97.74/15.67 89.24/0.309.53/10.16 89.84/2.13 63.96/0.19 (200 ul/mice) 4 9.76/10.59 92.51/15.5782.83/0.10 11.50/10.03 60.13/14.56 8.56/1.59 5 10.33/10.05 80.12/11.3470.98/0.30 11. Heptavalent-KLH + 10 ug 1 10.96/10.08 91.06/1.6455.98/0.02 9.76/10.81 26.58/2.13 5.42/0.16 QS-21 2 9.88/10.41 94.84/2.3272.07/0.02 9.80/11.58 88.16/22.37 34.36/0.94 (200 ul/mice) 3 11.0/10.5799.34/9.99 93.96/0.84 9.86/10.79 87.20/11.70 76.90/0.14 4 11.05/10.6588.58/3.78 61.69/0.13 10.07/10.45 71.56/3.79 26.42/0.15 5 10.86/10.2781.44/6.27 69.68/0.14 12. Heptavalent-KLH + 3 ug 1 18.54/11.2967.73/1.59 26.33/0.18 10.98/9.97 16.98/1.97 6.40/0.09 ER803022 29.69/9.60 74.76/0.72 33.45/0.08 10.62/10.33 36.76/2.05 3.73/0.07 (200ul/mice) 3 9.65/10.62 91.62/2.89 46.96/0.31 10.16/10.03 28.33/4.883.26/0.10 4 10.78/10.0 22.58/4.14 9.51/0.36 5 10.90/10.94 48.15/7.2910.64/0.23 13. Heptavalent-KLH + 10 ug 1 10.08/10.56 74.85/8.7140.57/0.24 10.96/10.20 64.39/3.85 7.58/0.17 ER803732 2 11.03/10.9136.35/5.07 4.79/0.07 9.84/10.19 3.67/11.73 0.47/0.73 (200 ul/mice) 310.22/10.54 65.94/30.22 7.87/0.63 10.83/10.07 66.06/25.19 8.66/0.82 410.75/10.07 91.69/12.15 22.96/0.41 5 10.87/11.04 35.45/6.88 1.38/0.1114. 30 ug KLH + 100 ug 1 9.42/11.35 13.72/9.84 10.08/2.29 10.85/10.495.49/4.48 3.29/2.53 GPI-100 2 12.14/11.31 17.50/2.09 6.41/0.010.58/10.44 10.57/3.53 14.38/0.21 (200 ul/mice) 3 9.82/10.97 1.64/1.050.13/0.02 9.19/11.45 15.10/1.03 11.63/0.01 4 11.20/11.84 13.25/2.1410.05/0.12 5 10.51/9.95 8.98/5.40 1.38/0.06 Heptavalent: Tn(c) (3 ug),TF(c) (3 ug), sTn(c) (3 ug), MUC1-1G5 (3 ug), Ley (10 ug), Globo H (10ug), and GM2 (10 ug). New GPI-100 used for all groups except group 9(batch J).

Third Series of Experiments

Polyvalent Conjugate Vaccine for Cancer

Tumor-specific antigens have been identified and pursued as targets forvaccines. The inventors' previous work has shown that monovalentvaccines utilizing the tumor antigens Globo H, Lewisy, GM2, glycosylatedMUC-1, Tn(c), sTn(c), or TF(c) conjugated to KLH to be safe with localerythema and edema but minimal systemic toxicities. As a result ofvaccination with these monovalent vaccines, most patients generatedspecific high titer IgM or IgG antibodies against the respectiveantigen-KLH conjugates. The present invention provides multivalentvaccines wherein the components of the monovalent vaccine are combinedand administered with an adjuvant as treatment for cancer.

Vaccines consisting of a combination of tumor antigens administered witha saponin immunological adjuvant QS-21 or GPI-0100. The antigens areglycosylated MUC-1-G5, Globo H, GM2, Le^(y), Tn(c), sTn(c), and TF(c).In each case the antigen is conjugated to Keyhole Limpet Hemocyanin(KLH).

The preferred ranges of the antigen and adjuvant doses are as follows:

-   -   Glycosylated MUC-1-G5: 0.1 to 30 g;    -   Globo H: 0.1 to 100 g;    -   GM2: 0.1 to 100 g;    -   Le^(y): 0.1 to 60 g;    -   Tn(c): 0.1 to 100 g;    -   sTn(c): 0.1 to 100 g;    -   TF(c): 0.1 to 30 g;    -   QS-21: 25-200 g;    -   GPI-0100: 1-25 mg.

Example 1 Comparison of the Immune Response After Immunization withMonovalent and Hexavalent-KLH Conjugate Vaccines Against Prostate Cancer

The immune response of the five initial patients receiving hexavalentvaccine with the immune responses of patients who had previously beenimmunized with the respective monovalent vaccines is compared in thefollowing five tables. Shown are the reciprocal mean peak ELISA titerfor IgM and IgG after immunization and FACS assay (% of positivecells/mean intensity) using the MCF-7 tumor cell line. The comparisonfor GM2-KLH is pending. Comparing the responses induced by monovalentand hexavalent vaccines, there was no significant difference in theantibody responses against any of the five antigens tested to date.Combination of six individual conjugates into a single vaccine does notsignificantly change the antibody response against the individualantigens.

Hexavalent Versus Monovalent: Prostate MCF-7 Patient Elisa (TF-HSA) FACSTrial Patient Sera IgM IgG IgM %/Mean 8.6.01 protocol 98-048 M1 week 110 10 10/37 TF(c)-KLH + QS21 week 7 1280+ 160 11/39 dosage: 1 ug week 91280 40 17/50 M2 week 1 10 0 10/21 week 7 1280++ 10 52/54 week 9 1280+++10 72/64 M3 week 1 0 0 11/135 week 7 1280++ 160 2/28 week 9 1280++ 12806/104 M4 week 1 0 0 10/32 week 7 160 10 18/43 week 9 160 160 26/47 M5week 1 0 0 10/36 week 7 320 0 9/28 week 9 320 0 8/22 protocol 00-064 H1week 1 20 0 11/64 Hexavalent Conjgate + QS21 week 7 1280 160 7/55TF(c)dosage: 3 μg week 12 1280 160 3/31 H2 week 1 40 10 9/37 week 7 1280160 21/50 week 12 640 160 17/43 H3 week 1 40 0 10/35 week 7 1280 2054/65 week 12 640 40 34/47 H4 week 1 80 0 10/26 week 7 1280 80 22/43week 12 1280 20 19/54 H5 week 1 0 0 10/13 week 7 1280 320 57/26 week 121280 320 47/22 Controls C1 8.27.99 1280 1280 8.10.01 protocol 98-002 M1week 1 0 80 10/22 Tn(c)-KLH + QS21 week 7 40 2560 7/18 dosage: 3 μg week9 80 2560 9/21 M2 week 1 10 80 11/26 week 7 320 5120 32/40 week 9 6405120 37/42 M3 week 1 20 40 11/25 week 7 640 640 12/26 week 9 1280 128013/27 M4 week 1 40 40 10/49 week 7 640 1280 9/40 week 9 1280 1280 8/39M5 week 1 10 80 11/25 week 7 1280 5120 12/26 week 9 1280 5120 13/27protocol 00-064 H1 week 1 80 0 11/64 Hexavalent Conjgate + QS21 week 7320 1280 7/55 Tn dosage: 3 μg week 12 320 1280 3/31 H2 week 1 320 109/37 week 7 1280 320 21/50 week 12 1280 320 17/43 H3 week 1 10 0 10/35week 7 320 640 54/65 week 12 640 640 34/47 H4 week 1 40 160 10/26 week 7640 5120 22/43 week 12 320 5120 19/54 H5 week 1 0 20 10/13 week 7 320640 57/26 week 12 320 640 47/22 Controls C2 1280+ 640 8.17.01 PROTOCOL96-055 M1 week 1 0 0 11/40 GloboH-KLH + QS21 week 7 20 0 9/30 dosage: 10μg week 9 20 0 9/40 M2 week 1 0 0 11/33 week 7 0 0 12/42 week 9 0 014/52 M3 week 1 20 0 week 7 640 0 week 9 640 0 M4 week 1 10 0 11/26 week7 160 0 19/39 week 9 160 0 25/39 M5 week 1 40 0 10/26 week 7 160 0 17/38week 9 off study off study PROTOCOL 00-064 H1 week 1 40 0 11/41Hexavalent Conjgate + QS21 week 7 160 0 9/36 GloboH dosage: 10 μg week12 160 80 6/27 H2 week 1 160 0 11/33 week 7 640 0 11/36 week 12 320 015/40 H3 week 1 40 0 11/29 week 7 20 0 56/59 week 12 20 0 43/46 H4 week1 80 0 10/34 week 7 160 0 21/46 week 12 80 0 16/44 H5 week 1 40 0 10/15week 7 80 0 64/35 week 12 80 0 45/27 Controls C3 week 26 640 C4 12808.31.01 PROTOCOL 00-075 M1 week 1 0 0 11/23 LewY-MMCCH-KLH week 7 0 013/26 dosage: 20 μg week 9 0 0 24/92 M2 week 1 0 0 10/25 week 7 0 020/32 week 9 0 0 14/24 M3 week 1 0 0 11/30 week 7 40 80 8/20 week 9 2040 14/41 M4 week 1 0 0 11/46 week 7 0 0 10/50 week 9 0 10 9/44 M5 week 10 0 10/50 week 7 40 0 3/30 week 9 0 10 18/63 PROTOCOL 00-064 H1 week 1 00 11/41 Hexavalent Conjgate + QS21 week 7 0 0 9/36 Ley dosage: 10 μgweek 12 0 0 6/27 H2 week 1 0 0 11/33 week 7 0 0 11/36 week 12 0 0 15/40H3 week 1 0 0 11/29 week 7 10 0 56/59 week 12 0 0 43/46 H4 week 1 0 1010/34 week 7 10 10 21/46 week 12 10 0 16/44 H5 week 1 0 0 10/15 week 7 00 64/35 week 12 0 0 45/27 Controls C5 2560 C6 640 8.19.01 PROTOCOL 99-23M1 week 1 0 0 10/25 MUC33G 5 site - KLH + QS21 week 7 160 640 17/31dosage: 3 μg week 9 160 640 31/45 M2 week 1 0 0 10/29 week 7 2560 64038/53 week 9 2560 640 35/43 M3 week 1 0 20 10/28 week 7 2560 160 36/57week 9 2560 320 43/60 M4 week 1 0 0 11/41 week 7 2560 80 12/35 week 9640 320 12/38 M5 week 1 0 0 11/30 week 7 40 0 61/65 week 9 40 80 59/67PROTOCOL 00-064 H1 week 1 0 0 11/41 Hexavalent Conjgate + QS21 week 7 20640 9/36 MUC33 dosage: 3 μg week 12 0 160 6/27 H2 week 1 0 0 11/33 week7 0 40 11/36 week 12 0 80 15/40 H3 week 1 0 0 11/29 week 7 160 160 56/59week 12 40 320 43/46 H4 week 1 0 20 10/34 week 7 40 320 21/46 week 12 40160 16/44 H5 week 1 0 0 10/15 week 7 40 160 64/35 week 12 0 80 45/27Controls C7 2560 640

Immunization of mice with Heptavalent-KLH Conjugates* plus GPI-100 orQS-21 Mean Value: Group # Antigen ELISA FACS MCF-7 LSC CDC(MCF-7) # LSCMice # Pre Post 1. 3 ug GM2- Pre Post Pre Post 1:200 Pre Post 1:200KLH + 100 ug IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgGIgM G3PI-100 Tn 10.43% 10.44% 9.40% 8.97% 6.47% 0.55% 10.59% 10.60%6.41% 1.92% 3.63% 0.22% (100 ul/mice) Tf sTn Muc1-G5 Ley globo H GM2 0 00 0 2. 3 ug GloboH- Pre Post Pre Post 1:200 Pre Post 1:200 Mice # PrePost KLH + 100 ug IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgGIgM IgG IgM GPI100 Tn 10.77% 10.57% 15.22% 22.50% 13.47% 2.78% 10.33%10.75% 7.08% 2.46% 4.70% 0.25% (100 ul/mice) Tf 10.08% 10.57% 8.01%32.59% 6.86% 7.39% sTn (mice#1 Muc1-G5 re-tested.) Ley globo H 0 0 6405120 GM2 3. 3 ug Ley-KLH + 100 ug Pre Post Pre Post 1:200 Pre Post 1:200Mice # Pre Post GPI-100 IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgMIgG IgM IgG IgM 3-1 5.94% 46.42% (100 ul/mice) Tn 11.15% 10.34% 67.27%34.02% 20.32% 1.43% 10.79% 9.63% 74.13% 40.76% 11.12% 2.26% 3-2 4.47%75.05% Tf 3-3 4.49% 9.21% sTn 3-4 22.5% 119.0% Muc1-G5 3-5 11.02% 61.7%Ley 0 0 640 2560 globo H GM2 4. 3 ug Muc-1G-5- Pre Post Pre Post 1:200Pre Post 1:200 Mice # Pre KLH + 100 ug IgG IgM IgG IgM IgG IgM IgG IgMIgG IgM IgG IgM IgG IgM IgG IgM 4-1 3.30% GPI-100 Tn 11.12% 10.12%94.77% 7.99% 86.54% 0.46% 10.25% 10.12% 7.93% 2.55% 3.44% 0.23% 4-26.85% (100 ul/mice) Tf 4-3 14.0% sTn 4-4 4.49% Muc1- 0 0 2560+ 1280 4-54.23% G5 Ley globo H GM2 5. 3 ug STn(c)- Pre Post Pre Post 1:200 PrePost 1:200 Mice # Pre KLH + 100 ug IgG IgM IgG IgM IgG IgM IgG IgM IgGIgM IgG IgM IgG IgM IgG IgM 5-1 6.83% GPI-100 Tn 10.12% 10.23% 11.88%2.04% 6.11% 0.16% 10.49% 8.64% 96.67% 12.86% 88.91% 0.38% 5-2 10.91%(100 ul/mice) Tf 5-3 6.36% sTn 0 0 5120 320 5-4 7.11% Muc1- 5-5 4.37% G5Ley globo H GM2 6. 3 ug Tf(c)- Pre Post Pre Post 1:200 Pre Post 1:200Mice # Pre KLH + 100 ug IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgMIgG IgM IgG IgM GPI-100 Tn 10.58% 10.67% 10.56% 4.92% 9.14% 0.14% 9.78%10.29% 6.37% 3.06% 2.74% 0.16% (100 ul/mice) Tf 0 0 5120 320 sTn Muc1-G5 Ley globo H GM2 7. 3 ug Tn(c)- Pre Post Pre Post 1:200 Pre Post 1:200Mice # Pre KLH + 100 ug IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgMIgG IgM IgG IgM GPI-100 Tn 0 0 5120+ 2560 10.48% 10.31% 15.21% 17.10%7.02% 0.79% 11.04% 10.42% 12.20% 5.11% 5.81% 4.66% (100 ul/mice) Tf sTnMuc1-G5 Ley globo H GM2 KLH 8. Heptavalent- # LSC Mice # Pre Post KLH +100 ug Pre Post Pre Post 1:200 Pre Post 1:200 8-1 0.75% 2.99% GPI100 IgGIgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM 8-2 1.0%12.63% (200 ul/mice) Tn 0 10 5120+ 320 10.32% 10.5% 91.61% 18.46% 80.34%0.59% 10.43% 10.54% 96.04% 39.25% 83.91% 0.59% 8-3 −2.23% 44.48% Tf 0 105120 320 10.63% 10.14% 98.92% 24.41% 85.27% 7.44% 8-4 −3.12% 24.77% sTn0 0 2560 640 (mice #1 8-5 7.65% 27.28% Muc1-G5 0 0 5120++ 80 re-tested)VK9 2.05% Ley 0 0 80 160 3S193 82.2% globo H 0 80 10 1280 anti-GM2 71.2%GM2 0 0 0 10 696 32.2% 9. Heptavalent- Pre Post Pre Post 1:200 Pre Post1:200 Mice # Pre Post KLH + 100 ug IgG IgM IgG IgM IgG IgM IgG IgM IgGIgM IgG IgM IgG IgM IgG IgM 9-1 −2.79% 34.02% GPI (old Tn 0 0 2560 4010.51% 10.55% 84.31% 9.89% 60.45% 0.48% 10.40% 10.21% 92.22% 16.68%63.24% 0.25% 9-2 2.96% 9.42% batch) Tf 0 0 5120 40 9-3 4.50% 11.41% (200ul/mice) sTn 0 0 320 160 9-4 6.74% 9.51% Muc1-G5 0 0 1280 0 9-5 4.01%39.91% Ley 0 0 40 320 3S193 97.83% globo H 0 80 0 320 B72.3 5.34% GM2 00 0 0 HMFG.1 3.01% 10. Heptavalent- Pre Post Pre Post 1:200 Pre Post1:200 Mice # Pre Post KLH + 100 ug IgG IgM IgG IgM IgG IgM IgG IgM IgGIgM IgG IgM IgG IgM IgG IgM 10-1 −6.29% −3.3% GPI-100 + Tn 0 0 5120+ 64010.50% 10.44% 90.55% 12.59% 75.43% 0.27% 10.67% 10.24% 85.22% 11.17%54.16% 0.74% polysorbate 80 Tf 0 0 5120 320 (200 ul/mice) sTn 0 0 640640 Muc1-G5 0 0 5120 80 Ley 0 0 0 320 globo H 0 160 20 1280 GM2 0 0 0 011. Heptavalent- Pre Post Pre Post 1:200 Pre Post 1:200 Mice # Pre PostKLH + 10 ug IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgGIgM 11-1 1.84% 10.93% QS-21 Tn 0 20 5120+ 160 10.75% 10.39% 90.96% 4.8%70.67% 0.23% 9.92% 10.74% 70.28% 8.33% 40.71% 0.29% 11-2 0.71% 3.20%(200 ul/mice) Tf 0 80 5120+ 640 11-3 1.31% 5.88% sTn 0 0 2560 320 11-4−1.58% 18.36% Muc1-G5 0 0 2560 160 11-5 10.28% 27.09% Ley 0 0 10 10globo H 0 80 40 640 GM2 0 0 0 0 12. Heptavalent- Pre Post Pre Post 1:200Pre Post 1:200 Mice # Pre Post KLH + 3 ug IgG IgM IgG IgM IgG IgM IgGIgM IgG IgM IgG IgM IgG IgM IgG IgM ER803022 Tn 0 0 1280 160 11.89%10.49% 60.97% 3.33% 25.38% 0.23% 10.42% 10.29% 29.79% 4.55% 5.65% 0.14%(200 ul/mice) Tf 0 0 1280 80 sTn 0 0 80 80 Muc1-G5 0 0 80 20 Ley 0 0 020 globo H 20 160 80 640 GM2 0 0 0 0 13. Heptavalent- Pre Post Pre Post1:200 Pre Post 1:200 Mice # Pre Post KLH + 10 ug IgG IgM IgG IgM IgG IgMIgG IgM IgG IgM IgG IgM IgG IgM IgG IgM ER803732 Tn 0 20 160 160 10.59%10.62% 60.86% 12.60% 15.51% 0.29% 10.50% 10.24% 45.30% 11.86% 5.60%0.60% (200 ul/mice) Tf 0 10 320 160 sTn 0 40 20 160 Muc1-G5 0 0 160 80Ley 0 0 0 80 globo H 20 80 80 320 GM2 0 0 0 0 14. 30 ug KLH + Pre PostPre Post 1:200 Pre Post 1:200 Mice # Pre Post 100 ug IgG IgM IgG IgM IgGIgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM GPI-100 Tn 0 0 0 160 10.62%11.08% 11.02% 4.10% 5.61% 0.45% 10.32% 10.68% 9.09% 2.47% 7.96% 0.73%(100 ul/mice) Tf 0 40 0 320 sTn 0 0 0 80 Muc1-G5 0 10 0 160 Ley 0 0 0 0globo H 0 160 10 640 GM2 0 0 0 160 KLH 0 10 5120++ 320

Immunization of mice with Heptavalent-KLH Conjugates* plus QS-21 or GPI100 with or without mAb 9H10 against CTLA-4. Mean Value: Group # AntigenELISA (mean) ELISA (median) FACS MCF-7 1. Heptavalent-KLH + 10 ug PrePost Pre Post Pre Post 1:200 QS21 IgG IgM IgG IgM IgG IgM IgG IgM IgGIgM IgG IgM IgG IgM

Tn 0 0 57440 640 0 0 25600 640 9.89% 9.78% 95.87% 77.77% 78.03% 16.09%AVERAGE Tf 0 0 10240++ 260 0 0 10240+ 160 9.39% 9.94% 96.40% 80.96%79.70% 8.16% MEDIAN sTn 0 0 3520 580 0 0 1280 320 +CONTROL Muc1-G5 0 01280 80 0 0 1280 80 VK-9 40.08% Ley 0 32 1920 2560 0 0 1280 1280 MLS12897.56% globo H 0 48 672 128 0 80 640 160 antiGM2 #### GM2 MBr-1 #### 2.Heptavalent-KLH + 100 ug Pre Post Pre Post Pre Post 1:200 GPI100 IgG IgMIgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM Tn 0 0 19200 840 0 025600 320 10.15% 10.36% 92.97% 79.87% 66.63% 28.35% Tf 0 0 10240+ 260 00 10240+ 160 10.10% 10.35% 95.99% 93.53% 69.88% 26.96% sTn 0 0 720 360 00 320 80 Muc1-G5 0 0 1520 80 0 0 2560 160 Ley 0 0 3920 680 0 0 5120 640globo H 0 0 320 176 0 0 80 160 GM2 3. Heptavalent-KLH + 100 ug Pre PostPre Post Pre Post 1:200 GPI-100(Lyophilized) IgG IgM IgG IgM IgG IgM IgGIgM IgG IgM IgG IgM IgG IgM Tn 0 0 32000 480 0 0 25600 320 10.29% 10.14%92.85% 62.94% 65.36% 18.34% Tf 0 0 10240+ 340 0 0 10240+ 320 10.20%10.23% 92.35% 58.56% 63.59% 14.46% sTn 0 0 1200 260 0 0 1280 320 Muc1-G50 0 1120 0 0 0 320 0 Ley 0 0 320 720 0 0 480 640 globo H 0 0 128 440 0 00 200 GM2 4. Heptavalent-KLH + 100 ug GPI100′ + Pre Post Pre Post PrePost 1:200 polysorbate80 IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgMIgG IgM Tn 0 0 38400 400 0 0 25600 160 10.14% 10.18% 91.34% 57.09%70.79% 6.70% Tf 0 0 10240+ 140 0 0 10240+ 160 10.22% 10.29% 90.80%61.70% 67.27% 4.98% sTn 0 0 1960 200 0 0 1280 320 Muc1-G5 0 0 4000 20 00 2560 0 Ley 0 0 1440 960 0 0 1280 640 globo H 0 40 100 224 0 40 120 160GM2 5. Heptavalent-KLH + 100 ug GPI100 Pre Post Pre Post Pre Post 1:200Cytoxan 25 mg/Kg(I.P.) Day-1 IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgGIgM IgG IgM Tn 0 0 61400 808 0 0 25600 640 9.92% 10.30% 89.55% 72.17%64.38% 12.43% Tf 0 0 10240++ 560 0 0 10240++ 640 10.19% 10.19% 90.87%70.90% 66.42% 14.32% sTn 0 0 520 480 0 0 640 320 Muc1-G5 0 0 3840 0 0 02560 0 Ley 0 0 480 200 0 0 0 80 globo H 0 0 32 540 0 0 0 400 6.Heptavalent-KLH + 100 ug GPI100 Pre Post Pre Post Pre Post 1:200 mAbCTLA-4 in vaccine IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgGIgM (100 ug/mice) Day 0 Tn 0 0 32000 250 0 0 25600 160 10.33% 10.08%90.59% 65.89% 62.40% 17.62% Day 7 & 14 no CTLA-4 Tf 0 0 10240+ 1040 0 010240+ 1280 10.26% 10.01% 91.50% 62.97% 63.04% 8.00% sTn 0 0 6240 180 00 1280 200 Muc1-G5 0 0 1950 40 0 0 2560 40 Ley 0 0 180 2600 0 0 40 40globo H 0 0 0 240 0 0 0 240 GM2 7. Heptavalent-KLH + 100 ug GPI100 PrePost Pre Post Pre Post 1:200 mAb CTLA-4 in vaccine IgG IgM IgG IgM IgGIgM IgG IgM IgG IgM IgG IgM IgG IgM (100 ug/mice) Day 0 & 7 Tn 0 0 320001600 0 0 25600 1280 10.05% 10.31% 90.31% 85.42% 67.29% 29.21% Day 14 noCTLA-4 Tf 0 0 10240+ 1920 0 0 10240+ 1280 9.64% 10.41% 90.01% 85.58%69.08% 21.26% sTn 0 0 2880 100 0 0 2560 80 Muc1-G5 0 0 6400 60 0 0 512080 Ley 0 0 32 208 0 0 0 160 globo H 0 0 32 220 0 0 0 240 GM2 8.Heptavalent-KLH + 100 ug GPI100 Pre Post Pre Post Pre Post 1:200 mAbCTLA-4 not in vaccine IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgMIgG IgM I.P. day-1, 0, 1 Tn 0 0 85333 40 0 0 51200+ 40 9.69% 10.15%91.29% 45.33% 63.00% 6.19% Tf 0 0 10240++ 370 0 0 10240++ 320 9.63%10.09% 90.20% 45.21% 50.05% 7.00% sTn 0 0 480 0 0 0 640 0 Muc1-G5 0 03413 0 0 0 2560 0 Ley 0 0 320 106 0 0 160 0 globo H 0 0 53 240 0 0 0 80GM2 9. Heptavalent-KLH + 100 ug GPI100 Pre Post Pre Post Pre Post 1:200Control mAb ROR-g2 IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgGIgM 100 ug/mice Tn 0 0 56320 640 0 0 51200 640 10.25% 10.13% 93.16%90.64% 73.15% 45.88% I.P. day-1, 0, 1 Tf 0 0 10240+++ 2720 0 0 10240+++2560 10.27% 10.21% 94.17% 95.54% 76.62% 30.38% sTn 0 0 3040 5200 0 01280 320 Muc1-G5 0 0 10240 160 0 0 10240 160 Ley 0 0 144 464 0 0 80 160globo H 0 0 112 848 0 0 80 640 GM2 10. Heptavalent-KLH + 100 ug GPI100Pre Post Pre Post Pre Post 1:200 100 ug/mice IgG IgM IgG IgM IgG IgM IgGIgM IgG IgM IgG IgM IgG IgM Tn 0 0 81920 320 0 0 51200 160 10.42% 9.98%88.03% 76.96% 67.02% 33.26% Tf 0 0 10240++ 920 0 0 10240++ 320 10.50%10.01% 87.97% 90.19% 64.58% 24.15% sTn 0 0 2240 1440 0 0 2560 160Muc1-G5 0 0 4160 40 0 0 2560 0 Ley 0 144 144 2640 0 160 80 320 globo H 00 0 224 0 0 0 160 GM2

Example 2

A Preclinical Study Comparing Approaches for Augmenting theImmunogenicity of a Heptavalent KLH-Conjugate Vaccine Against EpithelialCancers

Previously using a series of monovalent vaccines, we have demonstratedthat the optimal method for inducing an antibody response against cancercell-surface antigens is covalent conjugation of the antigens to keyholelimpet hemocyanin (KLH) and the use of a saponin adjuvant. Inpreparation for testing a polyvalent (heptavalent)-KLH conjugate vaccinein the clinic, we have tested the impact on antibody induction againstthe 7 antigens of several variables described by others to augmentimmunogenicity. We explore here the impact of approaches for decreasingsuppression of the immune response (low dose cyclophosphamide andanti-CTLA4 mAb), different saponin adjuvants (QS-21 and GPI-0100), anddifferent methods of formulation (lyophilization and use of polysorbate80). After two sets of experiments, these results are clear:

-   1) Immunization with the heptavalent-KLH conjugate vaccine induces    high titers of antibodies against Tn (median ELISA titer IgM/IgG    320/10,240), sTn (640/2560), TF (320/5120), MUC1 (80/20,480) and    globo H (1280/10), lower titers of antibodies against Lewis Y    (160/80) and only occasional antibodies against GM2.-   2) These antibodies reacted with the purified synthetic antigens by    ELISA, and with naturally expressed antigens on the cancer cell    surface by FACS.-   3) Neither decreasing suppression with low dose cyclophosphamide or    anti-CTLA4 mAb, nor changing the standard formulation by    lyophilization or use of polysorbate 80 had any impact on antibody    titers.-   4) The two saponin adjuvants were comparably potent at our standard    doses (QS-21 10 ug and GPI-0100 100 ug) but a third experiment    comparing higher doses is in progress.

The high titers of antibodies against this heptavalent vaccine and theinability of these additional approaches to further augment antibodytiters confirms that the combination of conjugation to KLH and use of asaponin adjuvant is sufficiently optimized for testing in the clinic.

There is a broad and expanding body of pre-clinical and clinical studiesdemonstrating that naturally acquired, actively induced, and passivelyadministered antibodies are able to eliminate circulating tumor cellsand micro metastases (1). Induction of antibodies against tumor antigensis more difficult than induction of antibodies against viral andbacterial antigens because most tumor antigens are normal or slightlymodified auto antigens and because actively growing tumors may set inmotion mechanisms which suppress the anti-cancer cell immune response.Consequently it may be necessary to overcome not only some level oftolerance but also some additional level of active suppression, makingthe immunization approach critical. We have previously reported that theoptimal approach for induction of antibodies against gangliosides and avariety of other carbohydrate and peptide antigens is covalentattachment of the tumor antigen to an immunogenic carrier molecule(keyhole limpet hemocyanin (KLH) was optimal (2,3)) plus the use of apotent immunological adjuvant. In our previous experience saponinadjuvants such as QS-21 and GPI-0100 were the optimal adjuvants (4,5).

In preparation for clinical trials with a heptavalent KLH-conjugatevaccine we test here the impact of several variables including 1)vaccine formulation (lyophilization or the use of polysorbate 80), 2)decreasing suppression (low dose cyclophosphamide or anti-CTLA4 mAb), or3) various doses of the two saponin adjuvants QS-21 and GPI-0100, onantibody titers against the individual antigens and tumor cellsexpressing these antigens.

Pathogen-free female BALB/c or C57BL/6 mice 6-10 weeks of age wereobtained from the Jackson Laboratory (Bar Harbor, Me.). QS-21 wasobtained from Aquila Biopharmaceuticals (Framingham, Mass. (nowAntigenics Inc., NYC, NY)), GPI-0100 was obtained from GalenicaPharmaceuticals, Inc. (Birmingham, Ala.). Cytoxan (25 mg/kg) waspurchased and injected IP one day prior to the first immunization. Thehybridoma for murine monoclonal antibody CTLA-4 was obtained from JimAllison (Berkeley, Calif.) and the mAb was prepared by Dr. Polly Gregor(MSKCC). The reactivity of mAb with CTLA-4 was confirmed. Polysorbate 80was purchased.

Immunization of mice: groups of five mice were immunized 3 times at oneweek intervals with the heptavalent vaccine containing 3 mcg of each ofthe 7 antigens covalently conjugated to KLH and mixed with GPI-0100 orQS-21 as indicated. Vaccines were administered subcutaneously over thelower abdomen. A 4th, booster, immunization was given at week 8.

Serological Assays: For the ELISA assay, glycosylated MUC1, globo H,Lewis Y or GM2, or Tn, sTn or TF conjugated to BSA, were coated on ELISAplates at an antigen dose of 0.1-0.2 mcg per well.

Phosphatase-conjugated goat anti-mouse IgG or IgM was added at adilution of 1:200 (Southern Biotechnology Associates, Inc., Birmingham,Ala.). Antibody titer was the highest dilution yielding absorbance of0.10 or greater.

FACS analysis: MCF-7 human breast cancer cells expressing all sevenantigens but especially Lewis Y and MUC1 and sTn, and LSC expressingespecially Lewis Y, sTn and Tn, were used. Single cell suspensions of5×10⁷ cells/tube were washed in PBS with 3% fetal calf serum andincubated with 20 mcl of full strength or 1/200 diluted antisera for 30minutes on ice. 20 microliters of 1/15 goat anti-mouse IgG or IgMlabeled with FITC were all added and percent positive cells and meanfluorescent intensity (MFI) of stained cells analyzed using a FACScan(Becton Dickenson, Calif.). Pre and post vaccination sera were analyzedtogether and the pre-treatment percent positive cells set at 10%.

Comparision of the Immune Response After Immunization with Monovalentand Hexavalent-KLH Conjugate Vaccines Against Prostate Cancer

Glycolipid and glycoprotein differentiation antigens such as GM2, GloboH, Lewis y, Tn, TF, and mucin 1 (MUC1) are over-expressed on the cellsurface of many tumors. Of the many approaches to immunization we havetested, covalent conjugation of antigens such as these to keyhole limpethemocyanin (KLH) plus the use of immunological adjuvant QS-21 has beenthe optimal approach for inducing IgM and IgG antibodies. Immunizationof patients with monovalent vaccines containing these antigens hasdemonstrated the consistent immunogenicity and safety of these vaccines.However, to overcome the heterogeneous nature of tumors, and of theimmune response in different individuals, we have recently vaccinated asmall group of patients (prostate cancer with rising PSA, but free ofdetectable disease) with a hexavalent-KLH vaccine containing GM2, GloboH, Le^(y), Tn(c), TF(c) and glycosylated MUC1 individually conjugatedwith KLH and mixed with immunological adjuvant QS-21. The main objectiveof this presentation is to compare the immune response of the sixinitial patients receiving hexavalent vaccine with the immune responsesof patients who had previously been immunized with the respectivemonovalent vaccines. All patients were vaccinated six times (weeks 1, 2,3, 7, 19 and 31) and bloods obtained pre treatment and on weeks 7 and 9were tested at one time. RECIPRICOL MEAN PEAK ELISA TITER AFTERIMMUNIZATION IgM/IgG.

Antigen GM2 Globo H Le^(y) Tn TF MUC1 Polyvalent Pending 160/0 0/0 640/640 1280/160  40/ vaccine  320 Individual Pending 160/0 0/101280/2560 1280/160 2560/ Vaccine  320

Because of the low response against Le^(y), we are continuing studiesaimed at creating a more immunogenic Le^(y) vaccine. Comparing theresponses induced by monovalent and hexavalent vaccines, there was nosignificant difference in the antibody responses against any of the fiveantigens tested to date. Combination of six individual conjugates into asingle vaccine does not significantly change the antibody responseagainst the individual antigens.

Experiment 1: Median ELISA titers and FACS results after vaccination ofgroups of 5 Balb/c mice with Heptavalent-KLH conjugate Group # AntigenELISA(mean) ELISA(median) FACS MCF-7 1. Heptavalent-KLH + 10 ug Pre PostPre Post Pre Post 1:200 QS21 IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgGIgM IgG IgM +CONTROL Tn 0 0 57440 640 0 0 25600 640 10% 10% 96% 78% 78%16% VK-9 40.08% Tf 0 0 10240++ 260 0 0 10240+ 160 9% 10% 96% 81% 80% 8%MLS128 97.56% sTn 0 0 3520 580 0 0 1280 320 antiGM2 98.87% Muc1- 0 01280 80 0 0 1280 80 MBr-1 58.06% G5 Ley 0 32 1920 2560 0 0 1280 1280globo H 0 48 672 128 0 80 640 160 GM2 2. Heptavalent-KLH + 100 ug PrePost Pre Post Pre Post 1:200 GPI100 IgG IgM IgG IgM IgG IgM IgG IgM IgGIgM IgG IgM IgG IgM Tn 0 0 19200 840 0 0 25600 320 10% 10% 93% 80% 67%28% Tf 0 0 10240+ 260 0 0 10240+ 160 10% 10% 96% 94% 70% 27% sTn 0 0 720360 0 0 320 80 Muc1- 0 0 1520 80 0 0 2560 160 G5 Ley 0 0 3920 680 0 05120 640 globo H 0 0 320 176 0 0 80 160 GM2 3. Heptavalent-KLH + 100 ugPre Post Pre Post Pre Post 1:200 GPI-100 IgG IgM IgG IgM IgG IgM IgG IgMIgG IgM IgG IgM IgG IgM (Lyophilized) Tn 0 0 32000 480 0 0 25600 320 10%10% 93% 63% 65% 18% Tf 0 0 10240+ 340 0 0 10240+ 320 10% 10% 92% 59% 64%14% sTn 0 0 1200 260 0 0 1280 320 Muc1- 0 0 1120 0 0 0 320 0 G5 Ley 0 0320 720 0 0 480 640 globo H 0 0 128 440 0 0 0 200 GM2 4.Heptavalent-KLH + 100 ug Pre Post Pre Post Pre Post 1:200 GPI100′ +polysorbate80 IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM Tn0 0 38400 400 0 0 25600 1601 10% 10% 91% 57% 71% 7% Tf 0 0 10240+ 140 00 10240+ 60 10% 10% 91% 62% 67% 5% sTn 0 0 1960 200 0 0 1280 320 Muc1- 00 4000 20 0 0 2560 0 G5 Ley 0 0 1440 960 0 0 1280 640 globo H 0 40 100224 0 40 120 160 GM2 5. Heptavalent-KLH + 100 ug Pre Post Pre Post PrePost 1:200 GPI100 IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgGIgM Cytoxan 25 mg/Kg Tn 0 0 61400 808 0 0 25600 640 10% 10% 90% 72% 64%12% (I.P.) Day-1 Tf 0 0 10240++ 560 0 0 10240++ 640 10% 10% 91% 71% 66%14% sTn 0 0 520 480 0 0 640 320 Muc1- 0 0 3840 0 0 0 2560 0 G5 Ley 0 0480 200 0 0 0 80 globo H 0 0 32 540 0 0 0 400 GM2 6. Heptavalent-KLH +100 ug Pre Post Pre Post Pre Post 1:200 GPI100 IgG IgM IgG IgM IgG IgMIgG IgM IgG IgM IgG IgM IgG IgM mAb CTLA-4 in Tn 0 0 32000 250 0 0 25600160 10% 10% 91% 66% 62% 18% vaccine Tf 0 0 10240+ 1040 0 0 10240+ 128010% 10% 92% 63% 63% 8% (100 ug/mice) Day 0 sTn 0 0 6240 180 0 0 1280 200Day 7 & 14 no Muc1- 0 0 1950 40 0 0 2560 40 CTLA-4 G5 Ley 0 0 180 2600 00 40 40 globo H 0 0 0 240 0 0 0 240 GM2 7. Heptavalent-KLH + 100 ug PrePost Pre Post Pre Post 1:200 GPI100 IgG IgM IgG IgM IgG IgM IgG IgM IgGIgM IgG IgM IgG IgM mAb CTLA-4 in Tn 0 0 32000 1600 0 0 25600 1280 10%10% 90% 85% 67% 29% vaccine Tf 0 0 10240+ 1920 0 0 10240+ 1280 10% 10%90% 86% 69% 21% (100 ug/mice) Day 0 & sTn 0 0 2880 100 0 0 2560 80 7 Day14 no CTLA-4 Muc1- 0 0 6400 60 0 0 5120 80 G5 Ley 0 0 32 208 0 0 0 160globo H 0 0 32 220 0 0 0 240 GM2 8. Heptavalent-KLH + 100 ug Pre PostPre Post Pre Post 1:200 GPI100 IgG IgM IgG IgM IgG IgM IgG IgM IgG IgMIgG IgM IgG IgM mAb CTLA-4 not in Tn 0 0 85333 40 0 0 51200+ 40 10% 10%91% 45% 63% 6% vaccine Tf 0 0 10240++ 370 0 0 10240++ 320 10% 10% 90%45% 50% 7% I.P. day-1, 0, 1 sTn 0 0 480 0 0 0 640 0 Muc1- 0 0 3413 0 0 02560 0 G5 Ley 0 0 320 106 0 0 160 0 globo H 0 0 53 240 0 0 0 80 GM2 9.Heptavalent-KLH + 100 ug Pre Post Pre Post Pre Post 1:200 GPI100 IgG IgMIgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM Control mAb ROR-g2 Tn 00 56320 640 0 0 51200 640 10% 10% 93% 91% 73% 46% 100 ug/mice Tf 0 010240+++ 2720 0 0 10240+++ 2560 10% 10% 94% 96% 77% 30% I.P. day-1, 0, 1sTn 0 0 3040 5200 0 0 1280 320 Muc1- 0 0 10240 160 0 0 10240 160 G5 Ley0 0 144 464 0 0 80 160 globo H 0 0 112 848 0 0 80 640 GM210.Heptavalent- Pre Post Pre Post Pre Post 1:200 KLH + 100 ug IgG IgMIgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM GPI100 Tn 0 0 81920 3200 0 51200 160 10% 10% 88% 77% 67% 33% 100 ug/mice 10240 10240+ Tf 0 0 ++920 0 0 + 320 11% 10% 88% 90% 65% 24% sTn 0 0 2240 1440 0 0 2560 160Muc1- 0 0 4160 40 0 0 2560 0 G5 Ley 0 144 144 2640 0 160 80 320 globo H0 0 0 224 0 0 0 160 GM2

Experiment 2: Median ELISA titers and FACS results against MCF-7 and LSCcells after vaccination groups of 5 C57BL/6 Mice with heptavalent-KLHconjugate vaccine Group # Antigen ELISA FACS withMCF-7 FACS with LSC #LSC Heptavalent- Pre Post Pre Post 1:200 Pre Post 1:200 KLH + IgG IgMIgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM 100 ug Tn 0 105120+ 320 10% 11% 91.61% 18.46% 80% 18% 10% 11% 96.04% 39.25% 84% 39%GPI100 Tf 0 10 5120 320 99% 24% 82% 21% (200 sTn 0 0 2560 640 ul/mice)Muc1- 0 0 5120++ 80 G5 Ley 0 0 80 160 globo 0 80 10 1280 H GM2 0 0 0 10Heptavalent- Pre Post Pre Post 1:200 Pre Post 1:200 KLH + IgG IgM IgGIgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM 100 ug Tn 0 0 256040 11% 11% 84.31% 9.89% 60% 10% 10% 10% 92.22% 16.68% 63% 17% GPI (oldTf 0 0 5120 40 batch) sTn 0 0 320 160 (200 Muc1- 0 0 1280 0 ul/mice) G5Ley 0 0 40 320 globo 0 80 0 320 H GM2 0 0 0 0 Heptavalent- Pre Post PrePost 1:200 Pre Post 1:200 KLH + IgG IgM IgG IgM IgG IgM IgG IgM IgG IgMIgG IgM IgG IgM IgG IgM 100 ug Tn 0 0 5120+ 640 11% 10% 90.55% 12.59%75% 13% 11% 10% 85.22% 11.17% 54% 11% GPI-100 + Tf 0 0 5120 320polysorbate sTn 0 0 640 640 80 (200 Muc1- 0 0 5120 80 ul/mice) G5 Ley 00 0 320 globo 0 160 20 1280 H GM2 0 0 0 0 Heptavalent- Pre Post Pre Post1:200 Pre Post 1:200 KLH + IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgGIgM IgG IgM IgG IgM 10 ug Tn 0 20 5120+ 160 11% 10% 90.96% 4.8% 71% 5%10% 11% 70.28% 8.33% 41% 8% QS-21 Tf 0 80 5120+ 640 (200 sTn 0 0 2560320 ul/mice) Muc1- 0 0 2560 160 G5 Ley 0 0 10 10 globo 0 80 40 640 H GM20 0 0 0 Heptavalent- Pre Post Pre Post 1:200 Pre Post 1:200 KLH + IgGIgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM 10 ug Tn 020 160 160 11% 11% 60.86% 12.60% 16% 13% 11% 10% 45.30% 11.86% 6% 12%ER803732 Tf 0 10 320 160 (200 sTn 0 40 20 160 ul/mice) Muc1- 0 0 160 80G5 Ley 0 0 0 80 globo 20 80 80 320 H GM2 0 0 0 0

Protocol 00-106: Pilot Phase Trial: Vaccination of Patients Who HaveOvarian, Fallopian Tube or Peritoneal Cancer with A PolyvalentVaccine-KLH Conjugate + QS-21 Vaccine: 10 ug GM2 10 ug Globo-H 10 ug LeY3 ug Muc1G5 3 ug Tn(c) 3 ug S-Tn(c) 3 ug TF(c) Patient Name Muc1-1G5Globo-H LeY GM2 Tn(c) S-Tn(c) TF(c) Patient # Vaccination Serology Sera# IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG Jun. 12, Jun.12, Jun. 27, Jun. 19, Jun. 21, Jun. 21, Jun. 24, Jun. 24, Jun 25, Jun.25 2002 2002 2002 2002 2002 2002 2002 2002 2005 2002 Patient 1 Jul. 3,Jul. 3, P7OVQ 2 0 0 0 0 0 0 0 0 0 20 2001 2001 Jul. 10, Jul. 31, P7OVQ 62560 640 40 0 0 0 0 20 1280 160 2001 2001 Jul. 17, Aug. 14, P7OVQ 9 2560640 40 0 0 0 0 20 1280 160 2001 2001 Aug. 14, Aug. 08, P7OVQ 15 2560 32040 0 0 0 0 20 1280 320 2001 2001 OFF TRIAL (+) Control M62 2560 640Positve control 2560 S193(1 mg/ml) 6400 Positve control 2560 Positvecontrol 640 Positve control 2560 Positve control 2560 2560 Positvecontrol 2560 Jun. 4, Jun. 4, Jun. 27, Jun. 26, 2002 2002 2002 2002Patient 2 Jul. 3 Jul. 3, P7OVQ 3 0 0 0 0 2001 2001 Jul. 10, Jul. 31,P7OVQ 7 80 20 0 0 2001 2001 Jul. 17, Aug. 14, P7OVQ 10 40 20 0 0 20012001 Aug. 14, Aug. 28, P7OVQ 13 20 40 0 0 2001 2001 Sep. 25, P7OVQ 20 2020 0 0 2001 Oct. 9, Oct. 9, P7OVQ 24 20 20 0 0 2001 2001 Oct. 23, P7OVQ30 20 20 0 0 2001 Jan. 11, P7OVQ 49 0 0 0 0 2002 Jun. 7, P7OVQ 65 0 02002 (+) Control Positve control 2560 640 Positve control 2560 Positvecontrol 640 Jun. 7, Jun. 7, Jun. 27, Jun. 26, 2002 2002 2002 2002Patient 3 Jul. 10, Jul. 10, P7OVQ 4 0 0 20 0 2001 2001 Jul. 17, Aug. 7,P7OVQ 8 1280 160 160 0 2001 2001 Jul. 24, Aug. 21, P7OVQ 12 320 40 80 02001 2001 Aug. 21, Sep. 4, P7OVQ 18 160 80 80 0 2001 2001 Oct. 2, P7OVQ22 80 40 40 0 2001 Oct. 16, Oct. 16, P7OVQ 26 40 40 80 0 2001 2001 Oct.30, P7OVQ 33 80 80 40 0 2001 Jan. 8, P7OVQ 47 40 40 80 0 2002 (+)Control M62 2560 1280 Positve control 2560 Positve control 1280 Jun. 10,Jun. 10, 2002 2002 patient 4 Jul. 24, Jul. 24, P7OVQ 5 0 0 2001 2001Jul. 31, Aug. 21, P7OVQ 14 1280 80 2001 2001 Aug. 7, Sep. 4, P7OVQ 171280 40 2001 2001 Sep. 4, Sep. 18, P7OVQ 23 160 20 2001 2001 Oct. 16,P7OVQ 27 160 20 2001 Oct. 30, Oct. 30, P7OVQ 32 80 40 2001 2001 Nov. 13,P7OVQ 36 20 20 2001 Jan. 22, P7OVQ 52 20 20 2002 Mar. 26, P7OVQ 63 20 202002 (+) Control M62 1280 640 Jun. 12, Jun. 12, Jun. 27, Jun. 19, Jun.21, Jun. 21, Jun. 24, Jun. 24, Jun. 25, Jun. 25, 2002 2002 2002 20022002 2002 2002 2002 2002 2002 patient 5 Aug. 16, Aug. 16, P7OVQ 11 0 0 00 0 0 0 0 0 0 2001 2001 Aug. 23, Sep. 27, P7OVQ 21 80 80 0 0 0 0 0 0 40160 2001 2001 Aug. 30, Oct. 11, P7OVQ 25 40 80 0 0 0 0 0 0 0 160 20012001 Sep. 27, OFF TRIAL 2001 (+) Control M62 2560 640 Positive control2560 S193(1 mg/ml) 6400 Positve control 2560 Positve control 640 Positvecontrol 2560 Positve control 2560 2560 Positve control 2560 Jun. 17,Jun. 17, 2002 2002 Patient 6 Aug. 30, Aug. 30, P7OVQ 16 0 0 2001 2001Sep. 6, Sep. 27, P7OVQ 19 160 20 2001 2001 Sep. 13, Oct. 25, P7OVQ 31 4020 2001 2001 Oct. 11, Nov. 22, P7OVQ 39 20 0 2001 2001 Dec. 6, Dec. 6,P7OVQ 41 20 0 2001 2001 Dec. 20, P7OVQ 44 20 20 2001 Mar. 8, P7OVQ 62 2020 2002 (+) Control M62 2560 320 Jun. 13, Jun. 13, Jun. 27, Jun. 19,Jun. 21, Jun. 21, Jun. 24, Jun. 24, Jun. 25, Jun. 25, 2002 2002 20022002 2002 2002 2002 2002 2002 2002 patient 7 Oct. 19, Oct. 19, P7OVQ 280 0 0 (re-do) 0 0 0 0 20 0 2001 2001 Oct. 26, Nov. 16, P7OVQ 35 640 40 0(re-do) 0 0 0 0 160 160 2001 2001 Nov. 2, Dec. 14, P7OVQ 42 80 20 0(re-do) 0 0 0 0 80 160 2001 2001 Nov. 30, Jan. 11, P7OVQ 48 80 20 0(re-do) 0 0 0 20 80 80 2001 2002 Jan. 25, Jan. 25, P7OVQ 53 80 20 0(re-do) 0 0 0 20 80 40 2002 2002 Feb. 8, P7OVQ 56 80 20 0 (re-do) 0 0 020 80 80 2002 Apr. 19, 2002 (+) Control M62 2560 160 Positve control2560 S193(1 mg/ml) 6400 Positve control 2560 Positve control 640 Positvecontrol 2560 Positve control 2560 2560 Positve control 2560 Jun. 18,Jun. 18, 2002 2002 Patient 8 Oct. 23, Oct. 23, P7OVQ 29 0 0 2001 2001Oct. 30, Nov. 20, P7OVQ 38 0 0 2001 2001 Nov. 6, Dec. 18, P7OVQ 43 0 02001 2001 Dec. 4, Jan. 15, P7OVQ 51 0 0 2001 2002 Jan. 29, Jan. 29,P7OVQ 54 40 0 2002 2002 OFF TRIAL (+) Control M62 2560 640 Jun. 18, Jun.18, 2002 2002 Patient 9 Nov. 6, Nov. 6, P7OVQ 34 0 0 2001 2001 Nov. 13,Dec. 4, P7OVQ 40 1280 80 2001 2001 Nov. 20, Jan. 1, P7OVQ 45 320 80 20012002 Dec. 18, Jan. 29, P7OVQ 55 320 80 2001 2002 Feb. 12, Feb. 12, P7OVQ58 160 40 2002 2002 Feb. 26, P7OVQ 60 160 80 2002 May 9, P7OVQ 64 40 802002 (+) Control M62 2560 320 Jun. 13, Jun. 13, Jun. 27, Jun. 19, Jun.21, Jun. 21, Jun. 24, Jun. 24, Jun. 25, Jun. 25, 2002 2002 2002 20022002 2002 2002 2002 2002 2002 Patient 10 Nov. 20, Nov. 20, P7OVQ 37 0 00 (re-do) 0 0 0 0 0 0 2001 2001 Nov. 27, Dec. 18, P7OVQ 46 320 80 320(re-do) 0 20 0 20 160 80 2001 2001 Dec. 4, Jan. 15, P7OVQ 50 320 320 320(re-do) 0 20 0 20 80 640 2001 2002 Jan. 1, Feb. 12, pl no show — — — — —— — — — — 2002 2002 Feb. 26, Feb. 26, P7OVQ 59 40 160 80 (re-do) 0 20 020 40 320 2002 2002 Mar. 12, pl no show — — — — — — — — — — 2002 May 21,pl no show — — — — — — — — — — 2002 (+) Control M62 2560 160 Positvecontrol 2560 S193(1 mg/ml) 6400 Positve control 2560 Positve control 640Positve control 2560 Positve control 2560 2560 Positve control 1280 Jun.12, Jun. 12, Jun. 27, Jun. 19, Jun. 21, Jun. 21, Jun. 24, Jun. 24, Jun.25, Jun. 25, 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 Patient11 Mar. 7, Mar. 7, P7OVQ 1 0 0 0 0 0 0 0 20 20 0 2001 2001 Feb. 5, Feb.5, P7OVQ 57 0 0 0 0 0 0 0 0 20 0 2001 2001 Feb. 12, Mar. 5, P7OVQ 61 020 0 0 0 0 0 0 40 80 2002 2002 Feb. 19, OFF TRIAL 2002 Mar. 19, 2002 (+)Control M62 2560 640 Positve control 2560 S193(1 mg/ml) 6400 Positvecontrol 2560 Positve control 640 Positve control 2560 Positve control2560 2560 Positve control 2560 *Patient received one vaccine beforeprotocol hold, restarted ~1 year later

Protocol # 01-019: Serological analysis of Breast cancer patientvaccinated with hexavalent vaccine ELISA GM2 ELISA MUC-1-1 ELISA LeYELISA Globo ELISA dOSM (10 mcg) 5G (3 mcg) Ceramide H Ceramide ELISA Tffor Tn (3 mcg) ELISA sTn Vaccine Sample (3/02) (03/02) (10 mcg)(04/02)(10 mcg)(05/02) (3 mcg)(05/02) (06/02) (3 mcg) Patient # Date DateSerology Top IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgMPatient 1 Jan. 16, Jan. 16, P7BRQ4 20 0 0 0 0 0 0 0 0 20 2002 2002 Jan.23, Jan. 23, P7BRQ9 0 0 0 0 0 0 0 0 0 40 2002 2002 Jan. 30, Jan. 30,P7BRQ11 0 0 0 0 0 0 0 20 0 40 2002 2002 Feb. 13, P7BRQ19 0 0 40 80 0 0 020 320 640 2002 Feb. 27, Feb. 27, P7BRQ28 0 0 20 80 0 0 0 0 80 1280 20022002 Mar. 13, P7BRQ37 0 40 40 80 0 0 0 20 160 1280 2002 Apr. 10, P7BRQ4480 320 0 0 0 20 80 1280 2002 May 22, May 22, P7BRQ62 40 640 2002 2002Jun. 5, P7BRQ67 2002 Controls >2560 320 (M62) 320 2560 (MCG170) 160 2560(LeYM12) 20 80/160 Mono Ab (S193) 1280 (GB81) 0 320 Mono Ab (VK9) 5120(P7BRQ17) 320 >2560 (slovin lab wk7) Patient 2 Jan. 16, Jan. 16, P7BRQ50 0 0 0 0 0 0 20 20 2002 2002 Jan. 23, Jan. 23, P7BRQ8 0 0 0 0 0 0 0 400 40 2002 2002 Jan. 30, Jan. 30, P7BRQ12 0 0 0 0 0 0 0 2560 160 640 20022002 Feb. 13, P7BRQ20 0 0 80 160 0 0 0 640 160 1280 2002 Feb. 27, Feb.27, P7BRQ27 0 80 40 20 0 0 0 640 160 1280 2002 2002 Mar. 13, P7BRQ36 0160 80 20 0 0 0 320 320 640 2002 Apr. 10, P7BRQ46 80 80 0 0 0 80 160 6402002 May 22, May 22, P7BRQ61 160 640 2002 2002 Jun. 05, P7BRQ68 2002Controls >2560 320 (P7BRQ17) >2560 (hexavalent) 80 320 (P7BRQ16) 640(LeYM12) 0 160 Mono Ab(S193) 1280 (GB81) 0 320 Mono Ab(VK9) 5120(P7BRQ17) 320 >2560 (slovin lab wk7) Patient 3 Jan. 7, Jan. 7, P7BRQ1 00 0 0 0 0 0 0 0 0 2002 2002 Jan. 14, Jan. 14, P7BRQ3 0 0 0 0 0 0 0 0 0 02002 2002 Jan. 21, Jan. 21, P7BRQ7 0 0 0 320 0 20 0 320 0 80 2002 2002Feb. 4, P7BRQ17 0 20 80 2560 0 40 0 640 320 2560 2002 Feb. 18, Feb. 18,P7BRQ23 0 0 80 640 0 20 0 320 320 1280 2002 2002 Mar. 4, P7BRQ30 0 0 80320 0 0 0 320 1280 640 2002 Apr. 1, P7BRQ41 80 80 0 0 0 80 320 320 2002May 13, May 13, P7BRQ57 0 20 160 320 2002 2002 May 27, P7BRQ63 0 20 2002Controls >2560 (P7BRQ17 320 (hexavalent) 160 1280 (P7BRQ16) 320 2560(LeYM12) 20 160 Mono Ab(S193) 1280 (GB81) 0 320/640 Mono Ab(VK9) 01280/2560 (P7BRQ17) 320 2560 (slovin lab wk7) ELISA GM2 ELISA MUC-1-1ELISA LeY ELISA Globo ELISA dOSM (10 mcg) 5G (3 mcg) Ceramide H CeramideELISA Tf for Tn (3 mcg) ELISA sTn Patient Vaccine Sample (3/02) (03/02)(10 mcg)(04/02) (10 mcg)(05/02) (10 mcg)(05/02) (06/02) (3 mcg) NameDate Date Serology Top IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgMIgG IgM Patient 4 Jan. 14, Jan. 14, P7BRQ2 0 0 0 0 0 40 0 20 0 40 20022002 Jan. 21, Jan. 21, P7BRQ6 0 0 0 0 0 20 20 80 0 20 2002 2002 Jan. 28,Jan. 28, P7BRQ10 0 0 0 80 0 40 0 320 20 160 2002 2002 Feb. 11, P7BRQ16 00 640 320 0 1280 0 0 640/1280 40 320 2002 Feb. 25, Feb. 25, P7BRQ26 0 0160 40 0 320 0 320 40 640 2002 2002 Mar. 11, P7BRQ34 0 0 160 20 0 640 80320 160 160 2002 did not Apr. 8, come in 2002 Apr. 15, P7BRQ47 320 80 0320 0 160 160 160 2002 May 20, May 20, P7BRQ58 0 40 320 160 2002 2002Jun. 3, P7BRQ66 80 40 2002 Controls >2560 (P7BRQ17) 320 (hexavalent)320 >2560 (P7BRQ16) 160 >2560 (LeYM12) 20 160 Mono Ab(S193) 1280 (GB81)20 320/640 Mono Ab(VK9) 0 1280/2560 (P7BRQ17) 320 >2560 (slovin lab wk7)Patient 5 Feb. 6, Feb. 6, P7BRQ15 0 0 0 0 0 0 0 0 0 0 2002 2002 Feb. 13,Feb. 13, P7BRQ22 0 0 0 20 0 0 0 0 0 0 2002 2002 Feb. 20, Feb. 20,P7BRQ25 0 0 0 0 0 0 0 0 0 0 2002 2002 Mar. 6, P7BRQ33 0 0 40 40 0 0 0 2020 40 2002 Mar. 20, Mar. 20, P7BRQ38 0 0 20 40 0 0 0 0 0 40 2002 2002Apr. 3, P7BRQ42 160 40 0 0 0 0 40 40 2002 May 1, P7BRQ51 0 20 20 20 2002Jun. 12, Jun. 12, P7BRQ71 2002 2002 Jun. 26, 2002 Controls >2560 320(P7BRQ17) 80 160 (hexavalent) 640 320 (P7BRQ16) 20 160 (LeYM12) 1280Mono Ab(S193) 0 320/640 (GB81) 0 1280/2560 Mono Ab(VK9) 320 >2560(P7BRQ17) 320 1280 (slovin lab wk7) Patient 6 Feb. 6, Feb. 6, P7BRQ14 020 0 0 20 0 0 0 0 0 2002 2002 Feb. 13, Feb. 13, P7BRQ18 0 20 0 0 0 0 0 00 0 2002 2002 Feb. 20, Feb. 20, P7BRQ24 0 40 0 80 0 0 0 160 160 80 20022002 Mar. 6, P7BRQ31 0 20 320 320 0 0 0 160 160 320 2002 Mar. 20, Mar20, P7BRQ39 0 20 160 320 0 0 0 80 40 160 2002 2002 Apr. 3, P7BRQ43 32040 0 0 0 40 160 160 2002 May 1, P7BRQ52 0 40 320 320 2002 Jun. 12, Jun.12, P7BRQ70 2002 2002 Jun. 26, 2002 Controls >2560 320 (P7BRQ17) 80 640(hexavalent) 640 320 (P7BRQ16) 0 80/160 (LeYM12) 1280 Mono Ab(S193) 0320/640 (GB81) 0 1280/2560 Mono Ab(VK9) 320 2560 (P7BRQ17) 320 1280(slovin lab wk7) ELISA GM2 ELISA MUC-1-1 ELISA LeY ELISA Globo ELISAdOSM (10 mcg) 5G (3 mcg) Ceramide H Ceramide ELISA Tf for Tn (3 mcg)ELISA sTn Patient Vaccine Sample (4/02) (04/02) (10 mcg)(04/02) (10mcg)(05/02) (10 mcg)(05/02) (06/02) (3 mcg) Name Date Date Serology TopIgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM Patient 7 Feb.27, Feb. 27, P7BRQ29 0 0 0 0 0 0 0 0 0 20 2002 2002 Mar. 6, Mar. 6,P7BRQ32 0 0 0 0 0 0 0 0 0 0 2002 2002 Mar. 13, Mar. 13, P7BRQ35 0 20 20320 0 0 0 0 320 160 2002 2002 Mar. 27, P7BRQ40 0 40 320 320 0 0 0 0640/1280 160 2002 Apr. 10, Apr. 10, P7BRQ45 0 80 320 640 0 0 0 0 640 802002 2002 Apr. 24, P7BRQ49 0 80 160 320 0 0 0 0 1280 80 2002 May 22,P7BRQ60 0 0 640 80 2002 Jul. 3, Jul. 3, 2002 2002 Controls >2560 320(P7BRQ17) 80 640 (hexavalent) 640 320 0 1280 (P7BRQ16) 0 160 (LeYM12)1280 Mono Ab(S193) 20 320 (GB81) 0 1280/2560 Mono Ab(VK9) 640 2560(P7BRQ17) 640 1280 (slovin lab wk7) (06/02) (06/02) (07/02) (07/02)Patient 8 Apr. 22, Apr. 22, P7BRQ48 2002 2002 Apr. 29, Apr. 29, P7BRQ532002 2002 May 6, May 6, P7BRQ54 2002 2002 May 20, P7BRQ59 2002 Jun. 3,Jun. 3, P7BRQ65 2002 2002 Jun. 17, P7BRQ72 2002 Jul. 15, 2002 Aug. 26,Aug. 26, 2002 2002 Sep. 9, 2002 Controls (P7BRQ17) (hexavalent)(P7BRQ16) (LeYM12) Mono Ab(S193) (GB81) Mono Ab(VK9) (P7BRQ17) (slovinlab wk7) Patient 9 Apr. 29, Apr. 29, P7BRQ50 2002 2002 May 6, May 6,P7BRQ55 2002 2002 May 13, May 13, P7BRQ56 2002 2002 May 27, P7BRQ64 2002Jun. 10, Jun. 10, P7BRQ69 2002 2002 Jun. 24, 2002 Jul. 22, 2002 Sep. 2,Sep. 2, 2002 2002 Sep. 16, 2002 Controls (P7BRQ17) (hexavalent)(P7BRQ16) (LeYM12) Mono Ab(S193) (GB81) Mono Ab(VK9) (P7BRQ17) (slovinlab wk7) Notes Monoclonals: Dilutions VK9 1:200; S193 1:400 DOSM Testedpositive by ELISA with Monoclonals 5F4 and HB-TN1. OSM Tested positiveby ELISA with Monoclonals 5F4, B72.3, and HB-TN1. Shaded region denotesPre sera (week 1) All samples were tested in duplicate. for patientsLewis and Smith, Lewis Y plates were coated at 0.3, future plates willbe coated at 0.2. Italics controls previously tested as positive.

Hexavalent Pilot study, protocol number #00-64, ELISA tested againstTF(c)-HSA Vaccination 1 2 3 4 5 Ag Patient Ab wkl 2 3 7 12 19 21 31 33TFC 1 Igm 40 40 40 40 >1280 40 40 40 40 HSA IgG 20 20 >1280 320 80 80 80160 320 2 IgM NMA 80 160 160 80 40 40 20 20 IgG 10 NMA 10 40 80 NMA NMA20 40 3 Igm 40 IgG NMA 4 IgM 40 20 320 80 80 80 NMA IgG NMA NMA 640 8040 160 320 5 IgM NMA 20 160 20 IgG 10 NMA 10 20 6 IgM 40 640 640 640 16080 80 160 IgG 640 >1280 >1280 >1280 >1280 >1280 >1280 >1280 7 IgM NMA 4080 640 160 80 40 80 80 IgG 10 320 NMA NMA 40 20 80 20 80 8 IgM NMA NMA160 160 IgG NMA NMA 10 80 9 IgM NMA NMA NMA 20 NMA NMA NMA NMA IgG NMANMA 40 NMA 640 80 >1280 160 10 IgM NMA 10 160 160 IgG NMA NMA 40 20 11IgM 20 IgG NMA 12 Igm 10 40 160 >1280 80 640 80 20 160 IgG NMA NMA NMANMA >640 20 40 40 80 13 IgM NMA IgG NMA 14 IgM 20 80 40 80 80 40 40 160160 IgG NMA 40 160 >1280 640 80 80 80 160 15 IgM NMA NMA 20 80 40 40 IgGNMA NMA 10 40 40 40 16 IgM 20 80 >1280 IgG 10 10 80 17 IgM NMA 10 NMA 4040 40 40 40 160 IgG NMA NMA 40 40 80 40 20 40 18 IgM 40 20 80 640 40 8080 20 80 IgG NMA NMA NMA 40 160 40 640 40 40 Positive Controls: IgM(19)1: 1280 IgG (7) 1: 640 (−) Human AB Serum As from Oct. 19, 2001 end pt.Titers: (+) IgM: 640 IgG: 640 (−) IgM: NMA IgG: NMA

Hexavalent Pilot study, protocol # 00-64 ELISA against glycosylatedMUC1-1 (5 sites) Vaccination 1 2 3 4 5 Ag Patient Number Ab wkl 2 3 7 1219 21 31 MUC 1 Igm NMA 40 >1280 >1280 >1280 >1280 >1280 >1280 1-1G5 IgGNMA NMA 80 160 >1280 640 >1280 >1280 32mer 2 IgM NMA NMA 640 10 NMA 1020 IgG NMA NMA 10 40 40 20.00 40 160 3 Igm 20 640 IgG NMA 40 4 Igm NMANMA 320.00 >1280 >1280 >1280 640 640 IgG NMA NMA 10 320160 >1280 >1280 >1280 5 IgM 10 40 >1280 >1280 IgG NMA NMA 160 >1280 6IgM NMA 320 >1280 320 320 160 80 IgG NMA 160 >1280 320 160 160 160 7 IgMNMA NMA 40 640 640 80 80 40 IgG NMA NMA640 >1280 >1280 >1280 >1280 >1280 8 IgM NMA NMA 160 IgG NMA NMA 10 9 IgMNMA NMA 40 160 80 20 20 10 IgG NMA NMA NMA 40 20 10 80 40 10 Igm NMA NMAIgG NMA NMA 11 IgM NMA NMA 10 40 20 160 160 80 IgG NMA NMA 10 320 160 4040 20 12 Igm 40 40 IgG NMA NMA 13 Igm NMA NMA 20 80 80 320 320 160 IgGNMA NMA 20 20 40 320 320 160 14 Igm 80 20 160 160 40 40 IgG NMA NMA NMANMA 40 20 15 Igm NMA NMA 640 640 640 10 10 10 IgG NMA NMA 640 640 640160 80 80 16 Igm NMA 20 IgG NMA 20 17 IgM NMA 20 320 160 40 640 320 80IgG NMA NMA 10 80 640 40 640 160 Positive Controls: (7) IgM 1: 2560 IgG1: 2560 Oct. 22, 2001 (−) AB Sera Controls as of Oct. 15, 2001: (+)Igm: >1280 IgG: >1280 (−) IgM: NMA IgG: NMA

Fourth Series of Experiments

Polyvalent Conjugate Vaccine for Cancer

Preliminary Data of Vaccination of High Risk Breast Cancer (BC) Patients(Pts) with a Heptavalent Antigen—Keyhole Limpet Hemocyanin (KLH)Conjugate Plus the Immunologic Adjuvant QS-21.

We have previously shown that following vaccination with single antigen(Ag)—KLH conjugates plus QS-21, the majority of BC pts generate specificantibody (AB) titers. (Clin Ca Res 6:1693, 2000; PNAS 98(6):3270, 2001;Proc ASCO 16:439a, 1997, 18:439a, 1999, 20:271a, 2001) Single Ag'stested have included MUC-1 (various peptide lengths), sTn clustered (c),GloboH and GM2. In an effort to improve and broaden the immune response,we treated BC pts with seven Ag's: 10 mcg each of GM2, GloboH, Lewisy;and 3 mcg each of TF(c), sTn(c), Tn(c) and glycosylated MUC-1, (32 aminoacid (aa) sequence, glycosylated at 5 sites per 20 aa tandem repeat).Each Ag was conjugated to KLH and mixed with 100 mcg of QS-21.Heptavalent vaccines were administered subcutaneously during weeks 1, 2,3, 7, and 19. We treated ten patients: median age 48 years (range 43-63yrs); Stage IV=3, Stage 2 with ≧4 positive nodes=7. Nine pts havecompleted immunization. Toxicity was limited to transient grade 2 localskin reactions and grade 1-2 flu-like symptoms. IgM and IgG AB titerswere considered positive for each antigen if there was at least aneightfold increase above baseline more than once, during weeks 1-19.Antibody responses are tabulated. (table) MUC1 and TF(c) seem mostimmunogenic. Flow cytometric analysis (FACS) was obtained pre and posttherapy to detect binding of IgM and IgG AB against MCF-7 tumor cells. Apositive FACS was defined as at least a threefold increase abovebaseline and was observed in 6/9 patients for IgM and 0/9 for IgG.Further analyses are ongoing. Our next cohort will evaluate the sameantigens conjugated to KLH but with GP-100 as the immunologic adjuvant.

Number of pts with positive AB response/Number of pts evaluable

Ag GM2 Ley MUC1 TF(c) sTn(c) Tn(c) GloboH IgM 2/9 1/9 8/9 8/9 4/9 7/96/9 IgG 0/9 1/9 8/9 8/9 1/9 0/9 0/9Objectives

-   -   Determine immune response against seven antigens and cell lines        expressing these antigens    -   Evaluate toxicity        Background    -   Preclinical data demonstrates that conjugation of an antigen        with keyhole limpet hemocyanin (KLH) and addition of the immune        adjuvant QS-21 augments immunogenicity (Cancer Immunol        Immunother 41:185, 1985; Cancer Res 56:3315, 1996)    -   Following vaccination with single antigen—KLH conjugates plus        QS-21, most breast cancer patients generated IgM and IgG        antibodies against the immunizing antigens (Clin Ca Res 6:1693,        2000; PNAS 98(6):3270, 2001; Proc ASCO 16:439a, 1997, 18:439a.        1999, 20:271a, 2001)    -   These single antigens have included MUC-1 (various peptide        lengths), sTn clustered (c), GloboH and GM2    -   To broaden the immune response, seven antigens were individually        conjugated to KLH and mixed with QS-21 to construct this        heptavalent vaccine        Vaccine Components    -   Antigens        -   Protein: glycosylated MUC-1 (32aa peptide)        -   Gangliosides: GM2, GloboH        -   Carbohydrates: Lewisy, sTn(c), Tn(c), TF(c)    -   Immunogenic protein carrier        -   KLH (by the following methods of conjugation):            -   MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester)                linker for TF(c), sTn(c), Tn(c), and MUC-1            -   MMCCH (4-[4-N-maleimidomethyl]cyclohexane-1-carboxyl                hydrazide) linker for GloboH and Le^(y)            -   Direct reductive amination for GM2    -   Immunologic Adjuvant        -   QS-21 (purified saponin fraction of tree bark)            Vaccine Components

Doses Antigens* GM2  10 mcg MUC-1  3 mcg LewisY  10 mcg GloboH  10 mcgTF(c)  3 mcg Tn(c)  3 mcg sTn(c)  3 mcg Adjuvant QS-21 100 mcg *(eachconjugated to KLH)Treatment and Evaluation Plan

WEEK # 1 2 3 5 7 9 13 19 21 (q 3 months) VACCINE 1 2 3 4 5 Blood √ √ √ √√ √ √ √ √ √ Samples for Immune ResponseEligibility Criteria

Breast cancer patients with any one of the following features:

-   -   Stage IV (stable on hormone therapy [tx])    -   Stage IV (no evidence of disease [NED])    -   Stage III    -   Stage II (≧4 positive nodes)    -   Ipsilateral breast or axillary recurrence    -   Rising CA15-3 or CEA levels and NED        Patient Characteristics    -   Total number of patients treated: 10        -   Total number of vaccinations completed: 50* (One patient was            delayed for unrelated issues)    -   Median age: 48 years (range 43-63 years)    -   Stage

II (with ≧ 4+ nodes): 7 IV (NED): 2 IV (stable on hormone tx): 1 n = 10Common Toxicities

-   -   Grade 1-2 injection site skin reactions        -   Grade 1-2-flu-like symptoms    -   No significant laboratory abnormalities        -   No definite autoimmune reactions            Response Criteria: ELISA    -   Serologic Response by ELISA (Enzyme-Linked Immunosorbent Assay)        -   IgM and IgG antibody titers were considered positive for            each antigen if there was a 2 eightfold increase above            baseline more than once, during weeks 1-19    -   Immunologic Response    -   Patient was considered a responder if there was a serologic        response to at least 3 of the 7 antigens        Response Criteria: FACS and CDC    -   Response by FACS (flourescence activated cell sorter) was        considered positive if there was the following increase above        baseline:        -   ≧3-fold increase in percent gated positivity, AND        -   ≧1.5-fold increase on MFI (mean flourescence intensity)    -   Response by CDC (complement-dependent cytotoxicity) was        considered positive if there was a 20% increase above baseline        Immune Response Data Elisa

GM2 MUC-1 Lewisy GLOBOH TF Tn sTn IgM IgG IgM IgG IgM IgG IgM IgG IgMIgG IgM IgG IgM IgG  1 − − + + − − − − + + − − − −  2 + − + + − − +− + + + − + −  3 + − + + − − + − + + + − + −  4 − − + + + − + + + + + −− −  5 − − − + − − − − − − − − − −  6 − − + + − − + − + + + − + −  7 +− + + − − − − + + + − − −  8 − − + + − − + − + + − − + −  9 − − + + −− + − + + − − − + 10 − − + + − − + − + + − − + − SUM 3 0 9 10 1 0 7 1 99 5 0 5 1Immune Response Data FACS and CDC

FACS (IgM) CDC Patient MCF-7 LSC Du-175 MCF-7 LSC  1 + + + − −  2 − +− + +  3 + − − − −  4 + + + + +  5 − − − − −  6 + + − + −  7 + + − − + 8 − + − + −  9 + + + − − 10 − − − + − SUM 6 7 3 5 3

CONCLUSION

-   -   Vaccination with a heptavalent antigen-KLH conjugate plus QS-21        is well tolerated in breast cancer patients    -   IgM and IgG antibody responses (to at least 3 of 7 antigens)        were observed in 8 patients and 2 patients respectively    -   MUC1 and TF(c) to be appear the most immunogenic of the seven        antigens in this vaccine    -   IgM antibody binding to tumor cells (MCF-7, LSC, Du-145) by FACS        analysis was observed in 6 patients, 7 patients, and 3 patients        respectively    -   There was no consistent evidence of IgG antibody binding to        tumor cells by FACS    -   There was evidence of CDC with the MCF-7 and LSC tumor cell        lines in 5 patients and 3 patients respectively    -   Our next cohort will evaluate the same antigens conjugated to        KLH but with GP-100 as the immunologic adjuvant

1. An immunogenic composition comprising (i) an adjuvant, and (ii) agroup of antigens comprising glycosylated MUC-1-G5, Globo H, GM2,Le^(y), Tn(c), sTn(c), and TF(c), wherein the antigens are eachindividually conjugated to a carrier molecule.
 2. The composition ofclaim 1, wherein the adjuvant is QS21.
 3. The composition of claim 1,wherein the adjuvant is GPI-0100.
 4. The composition of claim 1, whereinthe carrier molecule is Keyhole Limpet Hemocyanin (KLH).
 5. Thecomposition of claim 1, wherein the composition contains from about 0.1to about 30 ug of MUC-1-G5.
 6. The composition of claim 1, wherein thecomposition contains from about 0.1 to about 100 ug of Globo H.
 7. Thecomposition of claim 1, wherein the composition contains from about 0.1to about 100 ug of GM2.
 8. The composition of claim 1, wherein thecomposition contains from about 0.1 to about 100 ug of Tn(c).
 9. Thecomposition of claim 1, wherein the composition contains from about 0.1to about 100 ug of sTn(c)
 10. The composition of claim 1, wherein thecomposition contains from about 0.1 to about 30 ug of TF(c).
 11. Amethod for inducing antibody production in a subject bearing cancer thatexpresses one or more of the antigens contained in the composition ofclaim 1, comprising administering an effective amount of the compositionof claim 1 to the subject, wherein the cancer is selected from the groupconsisting of ovarian cancer, breast cancer, and prostate cancer.